ES2657628T3 - Polymeric conjugates containing acryloyloxyethylphosphorylcholine and its preparation - Google Patents

Abstract

A compound comprising a biologically active agent covalently bonded to a branched polymer containing phosphorylcholine, in which the polymer has two or more polymer arms extending from a single group, in which the polymer portion of the compound is poly [phosphate of 2- (methacryloxyethyl) -2'-5 (trimethylammonium) ethyl].

Description

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DESCRIPTION

Polymeric conjugates containing acryloyloxyethylphosphorylcholine and its preparation Cross-references to related applications Field of the invention

The present invention relates to polymeric reagents and conjugates thereof, methods for synthesizing polymeric reagents and conjugates, pharmaceutical compositions comprising the conjugates, and methods of using polymer conjugates that include therapeutic methods in which conjugates are administered to patients

Background of the invention

Efforts to formulate biologically active agents for administration should address a variety of variables that include the route of administration, the biological stability of the active agent and the solubility of the active agents in physiologically compatible media. The choices made in the formulation of biologically active agents and the selected routes of administration may affect the bioavailability of the active agents. For example, the choice of parenteral administration in the systemic circulation for biologically active proteins and polypeptides avoids the proteolytic environment found in the gastrointestinal tract. However, even when direct administration, such as by injection, of biologically active agents is possible, the formulations may be unsatisfactory for a variety of reasons, including the generation of an immune response to the administered agent and responses to any excipient, including burns. and bites Even if the active agent is not immunogenic and satisfactory excipients can be employed, the biologically active agents may have limited solubility and a short biological half-life that may require repeated administration or continuous infusion, which can be painful and / or inconvenient.

For some biologically active agents, a degree of success has been achieved in the development of suitable formulations of bioactive agents by conjugating the agents with water soluble polymers. The conjugation of biologically active agents with water soluble polymers is generally considered to provide a variety of benefits for the administration of biologically active agents, and in particular, proteins and peptides. Among the water soluble polymers employed, polyethylene glycol (PEG) has been more widely conjugated with a variety of biologically active agents, including biologically active peptides. A reduction in immunogenicity or antigenicity, increase in half-life, increase in solubility, decrease in clearance by the kidney and decrease in enzymatic degradation have been attributed to conjugates of a variety of water-soluble polymers and bioactive agents, including conjugates of PEG. As a result of these attributes, polymeric conjugates of the biologically active agent require less frequent dosing and may allow the use of less active agent to reach a therapeutic assessment criterion. Less frequent dosing reduces the total number of injections, which can be painful and require uncomfortable visits to health professionals.

Although some success has been achieved with the conjugation of PEG, the "PEGylation" of biologically active agents remains a challenge, since the conjugation of PEG can result in the loss of biological activity. A variety of theories have been advanced to explain the loss of biological activity after conjugation with PEG. These include blocking of sites necessary for the agent to interact with other biological components, either by the conjugation bond or by the agent that is buried within the PEG conjugate, particularly when the polymer is long and can "wrap" around apart from the active agent, thereby blocking access to potential ligands required for the activity.

Branched forms of PEG have been introduced for use in the preparation of conjugates to alleviate some of the difficulties encountered with the use of long and straight polymeric PEG chains. While the branched polymer can overcome some of the problems associated with conjugates formed with long linear PEG polymers, neither branched nor linear PEG polymer conjugates completely solve the problems associated with the use of conjugated bioactive agents. Both linear and branched PEG conjugates may, for example, suffer degradation rates that are too long or too short. A rapid degradation rate may result in a conjugate that has an in vivo half-life too short, while a too slow degradation rate may result in an unacceptable long conjugate half-life in vivo.

In view of the recognized advantages of conjugating bioactive agents with water soluble polymers, and the limitations of water soluble polymers, such as PEG, in the formation of conjugates suitable for therapeutic purposes, additional water soluble polymers are desirable to form conjugates. with bioactive agents. Water-soluble polymers, particularly those that have many of the advantages of PEG for use in conjugate formation, and which do not suffer from the disadvantages observed with PEG as a conjugation agent, would be desirable for use in the formation of agents. therapeutic and diagnostic. To this end, they are established

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2-methacryloxyethyl phosphorylcholine polymers for use in the preparation of conjugates of biologically active agents.

WO03 / 074090 describes comparable conjugates in which, however, the polymeric portion is ionic and further differs in its binding to the biological agent of the present invention.

Brief summary of the invention

The invention provides a compound comprising a biologically active agent covalently bonded to a polymer containing branched phosphorylcholine, wherein the plasmid has two or more polymeric arms extending from a single group, wherein the polymeric portion of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl phosphate].

In one embodiment the compound is a compound of formula (Ib) or a salt, hydrate, or isomer thereof: (A-Z- (Sp1) n) a-L- (K) b (Ib)

where

K is a group of the formula

image 1

in which the polymeric portion:

image2

of the compound is poly [2- (methacryloxyethyl) -2 '- (trimethylammonium) ethyl phosphate];

each occurrence of E is independently selected from the group consisting of halo and Nu, in which Nu represents a nucleophilic group;

Sp1 is a spacer group;

n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000; a is an integer that varies from 1-8; b is an integer that varies from 1-8;

L is selected from the group consisting of C1-4 alkyl, cycloalkyl, carboxy-C1-4 alkyl, carboxycycloalkyl, C1-4 alkoxy-C1-4 alkyl and cyclic alkyl ether;

A is a biologically active agent;

Z is the product of the reaction between a group present in said biologically active agent and a reactive group attached to said Sp1 group when n is 1, or a reactive group attached to L when n is 0; and * indicates the point of attachment of each group K to said L.

In another embodiment, the compound is a compound of formula (IIb), which are embodiments of compounds of formula (Ib) where a and b are both 1:

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image3

where

the polymeric portion:

image4

of the compound is poly [2- (methacryloxyethyl) -2 '- (trimethylammonium) ethyl phosphate];

E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), -N (C1-4 alkyl) 2, - OH, -O- (C1.4 alkyl), and -O- (C1-4 alkyl substituted with fluorine);

Sp1 is a spacer group;

n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000;

L is selected from the group consisting of C1-4 alkyl, cycloalkyl, carboxy-C1-4 alkyl, carboxycycloalkyl, C1-4 alkoxy-C1.4 alkyl and cyclic alkyl ether;

A is a biologically active agent; Y

Z is the product of the reaction between a group present in said biologically active agent and a reactive group attached to said Sp1 group when n is 1, or a reactive group attached to L when n is 0.

In another embodiment, the compound is a compound of formula (Illb), which are embodiments of compounds of formula (lb) where a = 2 and b = 1,

image5

in which the polymeric portion:

image6

of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate.

In another embodiment, the compound is a compound of formula (IVb), which are embodiments of compounds of formula (Ib) where a = 1 and b = 2,

image7

10 in which each polymeric portion:

image8

of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate.

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In another embodiment, the compound is a compound of formula (Vb), which are embodiments of compounds of formula (Ib) where a = 2 and b = 2,

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image9

in which each polymeric portion:

image10

of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate.

In another embodiment, the compound is a compound of formula (Ib) (IIb), (Illb) (IVb) or (Vb), where each polymeric portion:

image11

of the compound is poly [2- (methacryloxyethyl) -2 '- (trimethylammonium) ethyl phosphate];

each occurrence of E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), - N (C% 4 alkyl) 2, -OH, -O- (C1 alkyl. 4), and -O- (C1-4 alkyl substituted with fluorine);

each occurrence of Sp1 is independently selected from the group consisting of: -C12-12 alkyl-, -C3-12 alkyl-, - (C1-8 alkyl) - (C3-12 cycloalkyl) - (C0-8 alkyl) -, - (CH2) 1-12O-, (- (CH2) 1-6-O- (CH2) 1-6-) 1-12-, (- (CH2) 1-4-NH- (CH2) 1-4 ) 1-12, (- (CH2) 1-4-O- (CH2) 1-4) 1-12-O-, (- (CH2) 1-4-O- (CH2) 1-4-) 1 -12O- (CH2) 1-12-, - (CH2) 1-12- (C = O) -O-, - (CH2) 1-12-O- (C = O) -, - (phenyl) - (CH2) 1-3- (C = O) -O-, - (phenyl) - (CH2) 1-3- (C = O) -NH-, - (C1-6 alkyl) - (C = O) -O- (C0-6 alkyl) -, - (CH2) 1-12- (C = O) -O- (CH2) 1-12-, - CH (OH) -CH (OH) - (C = O ) -O- -CH (OH) -CH (OH) - (C = O) -NH-, -S-maleimido- (CH2) 1-6-, -S-maleimido- (C1-3 alkyl) - ( C = O) - NH-, -S-maleimido- (C1-3 alkyl) - (C5-6 alkyl) - (C0-3 alkyl) -, - (C1-3 alkyl) - (C5-6 alkyl) (C0-3 alkyl) - (C = O) -O-, - (C1-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -NH-, -S- Maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) -, - (Co-3 alkyl) -phenyl- (C = O) -NH-, - (CH2) 1-12-NH- (C = O) -, - (CH2) 1-12- (C = O) -NH-, - (phenyl) - (CH2) 1-3- (C = O) -NH-, -S- (CH2) - (C = O) -NH- (phenyl) -, - (CH2) 1-12- (C = O) -NH- (CH2) 1-12-, - ( CH2) 2- (C = O) -O- (CH2) 2-O- (C = O) - (CH2) 2- (C = O) -NH-, - (C1-6 alkyl) - (C = O) -N- (C1-6 alkyl) -, acetal, ketal, acyloxyalkyl ether, -N = Ch-, - (C1-e alkyl) -SS- (C1-6 alkyl) -, - (C1-a alkyl ) -SS- (C1-a alkyl) - (C = O) -O-, - (C1-6 alkyl) -SS- (C1-6 alkyl) - (C = O) -NH-, -SS- ( CH2) 1-3- (C = O) -NH- (CH2) 1-4-NH- (C = O) - (CH2) 1-3-, -SS- (C0-3 alkyl) - (phenyl) -, -SS- (C1-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) 1-5-, - (C1-3 alkyl) - (phenyl) - (C = O) - NH- (CH2) 1-5- (C = O) -NH-, -SS- (C1.3 alkyl) -, - (C1-3 alkyl) - (phenyl) - (C = O) -NH-, -O- (C1-C6 alkyl) -S (O2) - (C1-a alkyl) -O- (C = O) -NH-, -SS- (CH2) 1-3- (C = O) -, - (CH2) 1-3- (C = O) -NH-N = CSS- (CH2) 1-3- (C = O) -NH- (CH2) 1-5-, - (CH2) 1-3 - (C = O) -NH- (CH2) 1-5- (C = O) -NH-, - (CH2) 0-3-

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(heteroaryl) - (CH2) 0-3-, - (CH2) 0-3-phenyl- (CH2) 0-3-

image12

Y

n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000;

L is selected from the group consisting of C1-4 alkyl, cycloalkyl, carboxy-C1-4 alkyl, carboxycycloalkyl, C1-4 alkoxy-C1-4 alkyl and cyclic alkyl ether;

A is a biologically active agent;

Z is selected from the group consisting of: phosphate, phosphate ester, - (C = O) O-, carboxylic acid ester, thioester, amide, disulfide, amine, -NH (R1) -, amidine, hydrazone, -N = CH-, -NH-CH2-,

.R1

-N = C-

-NH-C (R1) H-, -S-CH2-C (OH) (R2) -, -O-CH2-C (OH) (R2) -, -C (= O) O-CH2-C ( OH) (R2) -, -NR2-CH2-C (OH) (R2) -, -S-CH2- C (SH) (R2) -, -O-CH2-C (SH) (R2) -, - C (= O) O-CH2-C (SH) (R2) -, -NR2-CH2-C (SH) (R2) -,

.R2

—N = C—

-C (R2) H-NH-, - (C = O) -NH- (C = O) -NH-,

R2

-C = N- (C = 0) -NH

-C (R2) H-NH- (C = O) -NH-, - (C = O) -NH- (C = O) -O-

R2

-C = N- (C = 0) -O

-C (R2) H-NH- (C = O) -O-, - (C = O) -NH- (C = S) -NH-,

R2

-C = N- (OS) -NH

-C (R2) H-NH- (C = S) -NH-, - (C = O) -NH- (C = S) -O-,

-C (R2) H-NH- (C = S) -O-,

C = N- (C = S) -0

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image13

-NH- (C = O) -NH-, -O- (C = O) -NH-, -NH- (C = S) -NH-, -O- (C = S) -NH-, -S -CH2CH2- (C = O) -, -S-CH2CH (CH3) - (C = O) - S- CH2CH2- (C = O) O-, -S-CH2CH (CH3) - (C = O) O-, -S-CH2CH2- (C = O) NH-, -S-CH2-CH2- (pyridyl) -, -S-CH2-CH2-SO2-, -S-CH2- CH2-SO2-, -S -CH2- (C = O) -O-, -S-CH2- (C = O) -NH-, -S-CH2- (C = O) -, and

image14

R1 is C1.6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms;

R2 is H, C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms; and with respect to compounds of formula (Ib),

K is a group of the formula

image15

the polymeric portion:

image16

of the compound is poly [2- (methacryloxyethyl) -2 '- (trimethylammonium) ethyl phosphate]; a is an integer that varies from 1-8; b is an integer that varies from 1-8;

* indicates the point of attachment of each group K to said L.

In any of the embodiments herein, the biologically active agent, A, may include a spacer group, (Sp2) p, as a means of binding the active agent A to Z, where Sp2 is selected from the group consisting of: -C1-12 alkyl-, -C3-12 cycloalkyl-, - (C1-a alkyl) - (C3-12 cycloalkyl) - (C0-8 alkyl) -, - (CH2) 1-12O-, (- (CH2 ) 1-6-O- (CH2) 1-6-) 1-12, (- (CH2) 1-4-NH- (CH2) 1-4) 1-12-, (- (CH2) 1-4 -O- (CH2) 1-4) 1-12-O-, (- (CH2) 1-4-O- (CH2) 1-4-) 1-12O- (CH2) 1-12, - (CH2K12 - (C = O) -O-, - (CH2) 1-12-O- (C = O) -, - (phenyl) - (CH2) 1-3- (C = O) -O-, - ( phenyl) - (CH2) 1-3- (C = O) -NH-, - (C1-6 alkyl) - (C = O) -O- (C0-6 alkyl) -, - (CH2) 1-12 - (C = O) -O- (CH2) 1-12-, -CH (OH) -CH (OH) - (C = O) -O - CH (OH) -CH (OH) - (C = O) -NH-, -S-maleimido- (CH2) 1- 6-, -S-maleimido- (C1-3 alkyl) - (C = O) -NH-, -S-maleimido- (C1-3 alkyl ) - (C5-6 cycloalkyl) - (C0-3 alkyl) -, - (C1-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -O-, - ( C1-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -Nh-, -S-maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) - , - (C0-3 alkyl) -f enil- (C = O) -NH-, - (CH2> m2-NH- (C = O) -, - (CH2) 1-12- (C = O) -NH-, - (phenyl) - (CH2 ) 1-3- (C = O) -NH-, -S- (CH2) - (C = O) -NH- (phenyl) -, - (CH2) 1-12- (C = O) -NH- (CH2> m2-, - (CH2) 2- (C = O) -O- (CH2) 2-O- (C = O) - (CH2) 2- (c = o) -NH-, - (alkyl C1-6) - (C = O) -N- (C1.6 alkyl) -, acetal, ketal, acyloxyalkyl ether, -N = cH-, - (C1-6 alkyl) -SS- (C0-6 alkyl) -, - (C1-6 alkyl) -SS- (C1-6 alkyl) - (C = O) -O-, - (C1-6 alkyl) -SS- (C1-6 alkyl) - (C = O) -NH-, -SS- (CH2) 1-3- (C = 0) -NH- (CH2> m- NH- (C = O) - (CH2) 1-3-, -SS- (C0- alkyl 3) - (phenyl) -, -SS- (C1-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) 1-5-, - (C1-3 alkyl) - (phenyl) - (C = O) -

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NH- (CH2) i-5- (C = O) -NH-, -SS- (C1.3 alkyl) -, - (Ci.3 alkyl) - (phenl) - (C = O) -NH -, -O- (Ci-C6 alkyl) -S (O2) - (Ci-6 alkyl) -O- (C = O) -NH-, -SS- (CH2) i-3- (C = O) -, - (CH2) i-3- (C = O) -NH-N = CSS- (CH2) i-3- (C = O) -NH- (CH2) i-5-, - (CH2) i -3- (C = O) -NH- (CH2) i-5- (C = O) -NH-, - (CH2) 0-3- (heteroaryl) - (CH2) 0-3-, - (CH2 ) 0-3-phenyl- (CH2) 0-3-,

image17

R2 R2

\ ‘_

- (C- | .fi alkyl) - (arl) -C = N (C ^ g alkyl) - - (Cj.6 alkyl) -C = N- (aryl) - (C! _ * alkyl) -

OR

C ^ alkyl - 0-P — O— (Co_g alkyl) OH

Y

wherein when p is 0, the spacer group Sp2 is not present and -Sp2- represents a covalent bond such that the biologically active agent "A" is directly linked to the group Z by one or more covalent bonds.

In any of the embodiments herein, the polymeric portion of the compounds is polyhydroxyethylmethacryloyl phosphorylcholine (poly-HEMA-PC).

In any of the embodiments herein, m is an integer that can be selected from the following ranges: 2-30, 30-i00, i00-500, 500-i,000 and i,000-2,000.

In any of the embodiments herein, n is i.

In any of the embodiments herein, n is 0 and - (Spi) ü- is a covalent bond.

In any of the embodiments herein, any of them: a = 2, and b = 3 or 4; or a = 3 or 4, and b = 2.

In any of the embodiments herein, said biologically active agent is selected from the group consisting of: drugs, vaccines, antibodies, antibody fragments, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (for example, mRNA, tRNA tRNA, RNAi, DNA, cDNA, antisense constructs, ribozymes, etc.).

In any of the embodiments herein, wherein said biologically active agent is selected from the group consisting of: agalsidase, alefacept, aspariginase, amdoxovir (DAPD), antide, becaplermin, botulinum toxin, including types A and B and lower molecular weight compounds with activity of botulinum toxin, calcitonins, cyanovirin, denileucine diftitox, erythropoietin (EPO) (i.e., i66 amino acid form and i65 amino acid form of human EPO), EPO agonists, alpha dornase, protein stimulant erythropoiesis (NESP), coagulation factors such as Factor V, Factor VII, Factor VlIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, von Willebrand factor; Ceredasa, cerezima, alpha-glucosidase, collagen, cyclosporine, alpha defensins, beta defensins, desmopressin, exendin-4, granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), alpha-i proteinase inhibitor, elcatonin, granulocyte and macrophage colony stimulating factor (GM-CSF), fibrinogen, filgrastim, growth hormones, human growth hormone (hGH), somatropin, growth hormone releasing hormone (GHRH), GRO-beta, GRO-beta antibody , bone morphogenic proteins such as bone morphogenic protein 2, bone morphogenic protein 6, OP-i; acid fibroblast growth factor, basic fibroblast growth factor, CD-40 ligand, heparin, human serum albumin, low molecular weight heparin (LMWH), alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, consensus interferon; interleukins and interleukin receptors such as interleukin-1 receptor, interleukin-2, interleukin-2 fusion proteins, interleukin-3 receptor antagonist, interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6 , interleukin-8, interleukin-i2, interleukin-i receptor 3, interleukin-i receptor 7; lactoferrin and lactoferrin fragments, luteinizing hormone releasing hormone (LHRH), insulin, pro-insulin, insulin analogues, amylin, C-peptide, somatostatin, somatostatin analogs, including octreotide, vasopressin, follicle stimulating hormone (FSH), imiglucerase, vaccine against influenza, insulin growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF), plasminogen activators such as alteplase, urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, and teneteplase; neural growth factor (NGF), osteoprotegerin, platelet-derived growth factor, tissue growth factors, transforming growth factor-i, growth factor

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vascular endothelial, leukemia inhibitor factor, keratinocyte growth factor (KGF), glial growth factor (GGF), T lymphocyte receptors, CD molecules / antigens, tumor necrosis factor (TNF), 1 monocyte chemoattractant protein, factors Endothelial growth, parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1, rasburicase, thymosin inhibitor alpha 1 IIb / IIIa, thymosin beta 10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin, compounds phosphodiesterase (PDE), VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates, respiratory syncytial virus antibody, cystic fibrosis transmembrane regulatory gene (CFTR), deoxyribonuclease (Dnase), bactericidal protein / permeability enhancer (BPI), and anti-CMV antibody. Examples of monoclonal antibodies include etanercept (a dimeric fusion protein consisting of the extracellular ligand binding portion of the 75 kD human TNF receptor bound to the Fc portion of IgG1), abciximab, adalimumab, afelimomab, alemtuzumab, antibody against B lymphocyte, atlizumab, basiliximab, bevacizumab, biciromab, bertilimumab, CDP-484, CDP-571, CDP-791, CDP-860, CDP-870, cetuximab, clenoliximab, daclizumab, eculizumab, edrecolomabolumab, epumaumabomabolumabiz, bizumabizumabibumaboluma, font , gemtuzumab ozogamicin, ibritumomab tiuxetan, infliximab, inolimomab, keliximab, labetuzumab, lerdelimumab, olizumab, lym-1 radiolabelled metelimumab, mepolizumab, mitumomab, muromonad-CD3, nebacumab, natalizumab, odulimomab, omalizumab, oregovomab, palivizumab, pemtumomab, pexelizumab, rhuMAb-VEGF, rituximab, satumomab pendetide, sevirumab, siplizumab, tositumomab, I131 tositumomab, trastuzumab, tuvirumab, visilizumab, and fragments and mimetics thereof.

In any of the embodiments herein, said biologically active agent is selected from the group consisting of: erythropoietin, granulocyte colony stimulating factor (G-CSF), alpha interferon, beta interferon, human growth hormone, and imiglucerase.

In any of the embodiments herein, said biologically active agent is selected from the group consisting of: tacrine, memantine, rivastigmine, galantamine, donepezil, levetiracetam, repaglinide, atorvastatin, alefacept, tadalafil, vardenafil, sildenafil, fosamprenavir, oseltamivir, valacyclovir and valganciclovir, abarelix, adefovir, alfuzosin, alosetron, amifostine, amiodarone, aminocaproic acid, aminohippurate sodium, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amlodipine, amsacrine, anagrelide, anastrozole, aprepitant, aripiprazole, asparaginase, atazanavir, atomoxetine, anthracyclines, Bexarotene, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, cabergoline, capecitabine, carboplatin, carmustine, chlorambucine, sodium cilastatin, cisplatin, cladribine, chlodronate, cyclophosphamide, cyproterone, reagetinoic acid, campttinoic acid, all-campttinoic acid, campttinoic acid, all-camptinocine, campttinoic acid, campttinoic acid, campttinoic acid, campttinoic acid, campttinoic acid ; dacarbazine, dactinomycin, daptomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, doxorubicin, dutasteride, eletriptan, emtricitabine, enfuvirtide, eplerenone, epirubicin, estramustine, ethinyl estradiol, etoposide, exemestane, ezetimibe, fentanyl, fexofenadine, fludarabine, fludrocortisone , fluorouracil, fluoximasterone, flutamide, fluticazone, fondaparinux, fulvestrant, gamma-hydroxybutyrate, gefitinib, gemcitabine, epinephrine, L-Dopa, hydroxyurea, icodextrin, idarubicin, ifosfamide, imatinib, itineconazole, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinotezolidene , levamisole, lisinopril, lovotiroxina sodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, memantine, mercaptopurine, mequinol, bitartrate metaraminol, methotrexate, metoclopramide, mexiletine, miglustat, mitomycin, mitotane, mitoxantrone, modafinil, naloxone, naproxen, nevirapine, nicotine , nilutamide, nitazoxanide, nitisinone, norethindrone, octreotide, oxaliplatin, palonosetron, pamidronate, pemetrexed, pergolide, pentostatin, pilcamicina, porfimer, prednisone, procarbazine, prochlorperazine, ondansetron, palonosetron, oxaliplatin, raltitrexed, rosuvastatin, sirolimus, streptozocin, pimecrolimus, sertaconazole, tacrolimus, tamoxifen, tegaserod, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, tiotropium, topiramate, topotecan, treprostinil, tretinoin, valdecoxib, celecoxib, rofecoxib, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, voriconazole, dolasetron, granisetron, formoterol, fluticasone, leuprolide, midazolam, alprazolam, amphotericin B, podophyllotoxins, nucleosidase antivirals, aroyl hydrazones, sumatriptan, eletriptan; macrolides such as erythromycin, oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin, flurithromycin, dithromromycin, josamycin, spiromycin, midecamycin, loratadine, desloratadine, leucomycin, mithyamycin, swithiamycin; fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin, amicacin, kanamycin, neomycin, and streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin, colistimethate; polymyxins such as polymyxin B, capreomycin, bacitracin, penems; penicillins, including penicillinase sensitive agents such as penicillin G, penicillin V; penicillinase resistant agents such as methicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, naphcillin; active agents of gram negative microorganisms such as ampicillin, amoxicillin, and hetacillin, cilia, and galampicillin; antipseudomonas penicillins such as carbenicillin, ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporins such as cefpodoxime, cefprozil, ceftbutene, ceftizoxime, ceftriaxone, cephalothin, cefapirin, cephalexin, cefradrine, cefoxitin, cephamandol, cefazolin, cephaloridin, cefaclorus, cephadroxyl, cephaloximatrin, cephaloximethyl, cephalophatrin cephaphthiophyte, cephalophatrin cephaphthiophyte cefoperazone, cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams such as aztreonam; and carbapenems such as imipenem, meropenem, and ertapenem, pentamidine isethionate, albuterol sulfate, lidocaine, metaproterenol sulfate, beclomethasone diprepionate, triamcinolone acetamide, budesonide acetonide, salmeterol, ipratrium trichlorate, chroma trichlorate of ergotamine; taxanes such as paclitaxel; SN-38 and thyrostost.

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In any of the embodiments herein, said biologically active agent is selected from the group consisting of: sodium aminohipurate, amphotericin B, doxorubicin, aminocaproic acid, aminolevulinic acid, aminosalicylic acid, metaraminol bitartrate, disodium pamidronate, daunorubicin, lovothyroxine, lovothyroxine, lovothyroxine, lovothyroxine, lovothyroxine, lovotyroxine lisinopril, cilastatin sodium, mexiletine, cephalexin, deferoxamine, and amifostine.

In any of the embodiments herein, said biologically active agent is a protein or polypeptide.

In any of the embodiments herein, said protein or polypeptide comprises an amino acid of synthetic origin.

The invention further provides a pharmaceutical composition comprising a compound of the invention as defined above. The compound may be a compound of formula (Ib), (IIb), (IIIb) (IVb) or (Vb) according to any of the above embodiments.

The pharmaceutical composition may further comprise a second biologically active agent.

The invention provides a method for increasing the biological half-life property of a biologically active agent, which comprises covalently binding a polymer containing branched phosphorylcholine to a biologically active agent, wherein the polymer has two or more polymer arms extending from a single group, in which the polymer portion of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate.

The phosphorylcholine-containing polymer is a branched polymer, each as understood by one skilled in the art.

The polymers of the invention (ie, polymers containing phosphorylcholine) can be of any useful size. In one embodiment, the polymers have a size greater than 0.5 kDa, for example, a size greater than 4 kDa. In one embodiment, the phosphorylcholine polymer has a molecular weight between about 0.5 kDa and about 800 kDa, for example, between about 0.5 kDa and about 400 kDa (for example, between about 0.5 kDa and about 200 kDa ). Polymers of the invention containing only two phosphorylcholine groups are contemplated. For example, polymers of the invention are contemplated containing two, three, four, five, six, seven and eight or more phosphorylcholine groups.

Many biologically active agents are described in the present application and, accordingly, the invention includes each of the biologically active agents described herein attached to the phosphorylcholine-containing polymers of the invention. The biologically active agents are bound to the phosphorylcholine-containing polymers of the invention by a covalent bond. Any covalent bond useful in the invention can be used. For example, and without limitation, biologically active agents can be linked to phosphorylcholine-containing polymers through a covalent bond of at least one of an amino group, a hydroxyl group, a sulfhydryl group and a carboxyl group of the biologically active agent. .

In one embodiment, the phosphorylcholine polymer covalently binds to a spacer and the biologically active agent covalently binds to the spacer. That is, in certain cases it may be useful to bind a biologically active agent to the phosphorylcholine-containing polymers of the invention using a spacer. For example, the covalently binding of a phosphorylcholinein-containing polymer to a biologically active agent through the use of a spacer can reduce the steric hindrance that prevents or reduces the effectiveness of the binding (i.e., the chemical bond reaction) that provides the coupling of the phosphorylcholine-containing polymer to the biologically active agent.

In a useful embodiment, the biologically active agent is a protein, that is, a therapeutic protein. In one embodiment, the biologically active agent is a human protein such as a human cytokine.

The biologically active agents of the invention can be obtained from any useful source and by any useful methodology. For example, the biologically active agents of the invention, such as human proteins (for example, human cytokines, such as erythropoietin, G-CSF, interferons, GM-CSF and human enzymes and human hormones) and antibodies, can be obtained by expression Heterologous gene in bacterial, yeast and cell cultures, such as mammalian cell cultures, insect cell cultures, plant cell cultures and avian cell cultures as understood in the art. The proteins of the invention, such as, human proteins (for example, human cytokines, such as erythropoietin, G-CSF, interferons, GM-CSF and human enzymes and human hormones) and antibodies, can also be obtained from such transgenic organisms such as transgenic birds (eg, transgenic chickens, transgenic quail and transgenic turkey), transgenic goats, transgenic cows and transgenic plants as understood in the art. It is also contemplated that human proteins for use as described herein can be produced by gene activation in human cell lines as understood in the art. Many of the biologically active agents can be obtained from natural sources or can be obtained from

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Organic synthesis reactions as understood in the art.

Brief description of the drawings

Figure 1 shows the amino acid sequence (165 amino acids) of human erythropoietin (EPO).

Figure 2 shows the amino acid sequence (174 amino acids) of the human granulocyte colony stimulating factor (G-CSF).

Figures 3 and 4 show an HPLC chromatogram illustrating the conjugation of an aldehyde functionalized polymer of the invention with G-CSF.

Figures 5, 6 and 7 show an HPLC chromatogram illustrating the conjugation of an aldehyde functionalized polymer of the invention with EPO.

Figure 8 shows an HPLC chromatogram illustrating the conjugation of an NHS functionalized polymer of the invention with G-CSF.

Figures 9 and 10 show an HPLC chromatogram illustrating the conjugation of an NHS functionalized polymer of the invention with EPO.

Figure 11 shows an HPLC chromatogram illustrating the conjugation of an NHS functionalized polymer of the invention with Interferon alfa.

Figure 12 shows an HPLC chromatogram illustrating the conjugation of an NHS functionalized polymer of the invention with G-CSF.

Figure 13 shows an HPLC chromatogram illustrating the conjugation of an NHS functionalized polymer of the invention with somatostatin.

Figure 14 shows an HPLC chromatogram illustrating the conjugation of an aldehyde functionalized branched polymer of the invention with EPO.

Detailed description of the invention

A. Introduction

For the purpose of the present invention, the following terminology will be used in accordance with the definitions set forth below.

"Water soluble polymer" refers to a polymer that is soluble in water at room temperature. A solution of a water-soluble polymer can transmit at least about 75%, more preferably at least about 95% of the light, transmitted by the same solution after filtration. On a weight basis, a water soluble polymer or segment thereof can be at least about 35%, at least about 50%, about 70%, about 85%, about 95% or 100% ( by weight of dry polymer) soluble in water.

The molecular weight in the context of the polymer can be expressed as an average molecular weight in number or an average molecular weight by weight. Unless otherwise indicated, all references to molecular weight herein refer to weight average molecular weight. Both molecular weight determinations, average in number and average in weight, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods can also be used to measure molecular weight values, such as the use of titration criteria analysis, or the measurement of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure ) to determine the number average molecular weight, or the use of light scattering, ultracentrifugation or viscosimetry techniques to determine the weight average molecular weight. The polymeric reagents of the invention are typically polydispersed (i.e., the number average molecular weight and the weight average molecular weight of the polymers are not equal), which possess low polydispersity values of preferably less than about 1.5, according to It is judged by gel permeation chromatography. In other embodiments, the polydispersities may be in the range of about 1.4 to about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, even more preferably less than about 1.05 , and much more preferably less than about 1.03.

The phrase "one" entity as used herein, refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or "a"), "one or more" and "at least one" may be used interchangeably herein.

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The term "approximately", as used herein, means the variation that could be seen in measurements taken between different instruments, samples and sample preparations.

The term "compound" as used herein is intended to include not only the specified molecular entity but also its pharmaceutically acceptable pharmacologically active derivatives, including, but not limited to, salts, conjugates of prodrugs such as esters and amides, metabolites, hydrates, solvates and the like.

The terms "protected", "protected form", "protection group" and "protective group" refer to the presence of a group (ie the protective group) that prevents or blocks the reaction of a chemically reactive functional group in particular in a molecule under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group that is protected, as well as the reaction conditions that will be employed and the presence of additional reactive or protective groups in the molecule, if any. The person skilled in the art will recognize protective groups known in the art, such as those found in the Treaty of Greene et al., "Protective Groups In Organic Synthesis," 3rd Edition, John Wiley and Sons, Inc., New York , 1999.

The terms "spacer" and "spacer group" are used interchangeably herein to refer to an atom or a collection of atoms optionally used to bond interconnecting moieties such as an end of a water soluble polymer and a reactive group of a biologically active agent and a reactive group. A spacer may be hydrolytically stable or may include a hydrolytically susceptible or enzymatically degradable bond.

"Alkyl" refers to linear or branched hydrocarbon chains. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl (i.e., 2-pentyl), 1-ethylpropyl (i.e., 3-pentyl), 3- methylpentyl, and the like. The term "alkyl" is also used to indicate a hydrocarbon group that may be linear or branched that may have two or more functionalities attached; and it is understood that "alkyl" includes alkylene when two functionalities are added. As used herein, "alkyl" does not include cycloalkyl unless expressly stated otherwise. When the alkyl groups may have a size range, that size range may be indicated by indicating the number of carbon atoms present in the alkyl group (for example, C1-3 or C1 to C3 alkyl for an alkyl group containing from one to three carbon atoms). In addition, when a range includes the value of "0" (for example, C0-3 alkyl), the group is not present in one embodiment, and if it intervenes between two groups, it constitutes a covalent bond.

The term "alkoxy" refers to the above alkyl groups attached to oxygen.

The "polymer containing poly (acryloyloxyethylphosphorylcholine)" represents an acrylic acid polymer containing at least one acryloyloxyethyl phosphorylcholine monomer such as 2-methacryloxyethyl phosphorylcholine (ie, 2-methacryloyl-2'-trimethylammonium-ethyl phosphate).

The term "carboxyalkyl" means an alkyl group (as defined herein) substituted with a carboxy group. The term "carboxycycloalkyl" means a cycloalkyl group (as defined herein) substituted with a carboxy group. The term "alkoxyalkyl" means an alkyl group (as defined herein) substituted with an alkoxy group. The term "carboxy" used herein refers to carboxylic acids and their esters.

Terms such as "haloalkyl," and "haloalkoxy" are intended to include monohaloalkyl (oxy) and polyhaloalkyl (oxy). For example, the term "C1-C6 haloalkyl" is intended to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

"Fluorine-substituted alkyl" refers to an alkyl group in which one, some, or all of the hydrogen atoms have been replaced by fluorine.

"Cytokine" in the context of this invention is a member of a group of protein signaling molecules that can participate in cell-cell communication in immune and inflammatory response. Cytokines are typically small water soluble glycoproteins that have a mass of approximately 8-35 kDa.

"Therapeutic proteins" are peptides or proteins that include an amino acid sequence that in whole or in part constitutes a drug and can be used in human or animal pharmaceutical applications. Those skilled in the art know numerous therapeutic proteins that include, without limitation, those described herein.

The "phosphorylcholine-containing polymer" is a phosphorylcholine-containing polymer. It is specifically contemplated that in each case where a phosphorylcholine-containing polymer is specified in this application for a particular use, a single phosphorylcholine may also be employed in said use.

"Cycloalkyl" refers to a cyclic hydrocarbon group containing from about 3 to 12, from 3 to 10, or from 3 to

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7 endocyclic carbon atoms. Cycloalkyl groups include fused, bridged and spiro ring structures.

The term "endocyclic" refers to an atom or group of atoms that comprises part of a cyclic ring structure.

The term "exocyclic" refers to an atom or group of atoms that are attached but do not define the cyclic structure of the ring.

"Cyclic alkyl ester" refers to a 4 or 5-membered cyclic alkyl group having 3 or 4 endocyclic carbon atoms and 1 endocyclic oxygen or sulfur atom (eg, oxetane, thietane, tetrahydrofuran, tetrahydrothiophene); or a 6 to 7 membered cyclic alkyl group having 1 or 2 endocyclic oxygen or sulfur atoms (eg, tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithian, 1,4 -ditiano, 1,4-oxatiano).

"Alkenyl" refers to an unsaturated linear or branched hydrocarbon group containing at least one carbon-carbon double bond. Non-limiting examples of alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and the like.

"Aryl" refers to a cyclic aromatic group having one or more aromatic rings, each having 5 or 6 endocyclic atoms independently selected from carbon, oxygen, nitrogen and sulfur. The aryl rings may be condensed. Non-limiting examples of aryl groups include naphthyl and phenyl. As used herein, "aryl" includes carbocyclic aryl, in which all the endocyclic atoms of the aromatic group are carbon atoms and heteroaryl groups.

"Heteroaryl" refers to an aryl group having one or more heteroatoms (for example, nitrogen, oxygen and / or sulfur) in at least one ring. Non-limiting examples include pyrrole, furanyl, thienyl, pyridyl, oxazolyl, thiazolyl, benzofuranyl, and benzothienyl.

"Electrophile" refers to an ion or atom or set of atoms, which can be ionic, which has an electrophilic center, that is, an electron-seeking center, capable of reacting with a nucleophile. An electrophile (or electrophilic reagent) is a reagent that forms a bond with its reaction partner (the nucleophile) by accepting both bond electrons from that reaction partner.

"Nucleophile" refers to an ion or atom or set of atoms, which can be ionic, that has a nucleophilic center, that is, a center that looks for an electrophilic or electrophilic center. A nucleophile (or nucleophile reagent) is a reagent that forms a bond with its reaction partner (the electrophile) by donating both bond electrons. A "nucleophile group" refers to a nucleophile after it has reacted with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, haloalkoxy and the like.

"Phosphorylcholine," also called "PC," refers to

0

* - O— P

one

OR

where * represents the junction point. Phosphorylcholine is a zwitterionic group and includes salts (such as internal salts), and protonated and deprotonated forms thereof.

"Reactive group" refers to a functional group that is capable of forming a covalent bond consisting of one or more bonds to a biologically active agent. Non-limiting examples include those illustrated in Table 1.

"Maleimido" refers to a pyrrole-2,5-dione-1-yl group having the structure

image18

image19

which after reaction with a sulfhydryl (for example, a thioalkyl) forms a -S-maleimido group having the structure

image20

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where it indicates the point of attachment of the maleimido group and indicates the point of union of the sulfur atom of the thiol to the rest of the original sulfhydryl carrier group.

A "hydrolytically susceptible bond" or "hydrolytically susceptible bond" refers to a chemical bond or bond, which can be a covalent bond that is hydrolyzed under physiological conditions. The tendency of a bond to hydrolyse may depend not only on the general type of bond that connects two central atoms between which the bond is cut, but also on the substituents attached to these central atoms. Non-limiting examples of hydrolytically susceptible bonds include esters of carboxylic acids, phosphate esters, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, and some amide bonds.

An "enzymatically degradable binding" means a bond that is subject to degradation by one or more enzymes.

Some hydrolytically susceptible bonds can also be enzymatically degradable. For example, esterases can act on carboxylic acid esters or phosphate esters, and proteases can act on peptide bonds and some amide bonds.

The terms "active agent" and "biologically active agent" are used interchangeably herein and are defined to include any agent, drug, compound or mixture thereof that provides any local or systemic physiological or pharmacological effect that can be demonstrated in vivo. or in vitro Non-limiting examples include drugs, vaccines, antibodies, antibody fragments, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, TRNA tRNA, RNAi, DNA, cDNA, antisense constructs, ribozymes, etc.).

The term "solvate" as used herein, means a compound of the invention or a salt thereof, which also includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces in an amount greater than about 0.3% when prepared according to the invention.

The term "hydrate" as used herein, means a compound of the invention or a salt thereof, which also includes a stoichiometric or non-stoichiometric amount of water joined by non-covalent intermolecular forces. Hydrates are formed by the combination of one or more water molecules with one of the substances in which the water retains its molecular state as H2O, said combination being able to form one or more hydrates.

For the purposes of this description, "isomers" refers to certain compounds of the present invention that possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers (for example, separate enantiomers). All of these are included by the term "isomers" within the scope of the present invention.

For the purposes of this description, "naturally occurring amino acids" found in proteins and polypeptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid , L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and / or L-valine. The "synthetic amino acids" found in proteins are any amino acid other than those mentioned as naturally occurring amino acids. Amino acids of synthetic origin include, without limitation, the D isomers of naturally occurring amino acids and mixtures of D and L isomers of naturally occurring amino acids. Other amino acids, such as 4-hydroxyproline, desmosin, isodesmosin, 5- hydroxylysine, epsilon-N-methyl-lysine, 3-methylhistidine, although found in naturally occurring proteins, are considered amino acids of synthetic origin found in proteins for the purpose of this description, since they are generally introduced by means other than ribosomal translation of mRNA.

"Linear" in reference to the geometry, architecture or general structure of a polymer, refers to the main chain derived from a single polymer monomer.

"Branched", in reference to the geometry, architecture or general structure of a polymer, refers to a polymer having 2 or more polymer "arms" extending from a single group, such as a group L that can be derived from a initiator used in a radical polymerization reaction by atomic transfer. A branched polymer may have 2 polymer arms, 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 8 polymer arms or more. For the purposes of this description, compounds that have three or more polymer arms that extend from a single linear group are referred to as having a "comb" structure or "comb" architecture.

"Bifurcated" refers to a multifunctional chemical structure in which multiple functional groups (reactive groups or biologically active agents) are joined in addition to one or more polymer arms (directly or through

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one or more atoms) to a chemical group such as an L group that can be derived from an initiator used in an atomic transfer radical polymerization reaction.

"Pharmaceutically acceptable" or "pharmaceutical composition" composition refers to a composition comprising a compound of the invention and a pharmaceutically acceptable excipient or pharmaceutically acceptable excipients.

The term "salt" includes, without limitation, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulfates, hydrogen sulfates, alkylsulfonates, arylsulfonates, acetates, benzoates, citrates, maleates, smokers, succinates, lactates, and tartrates; salts of alkali metal cations such as salts of organic amines of Na +, K +, Li + (eg, NaCl, KCl) or salts of alkaline earth metals such as salts of Mg or Ca.

"Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to an excipient that can be included in the compositions of the invention and that does not cause any significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, Ringer lactate, normal sucrose, normal glucose and the like.

"Patient" or "subject in need" refers to a living organism that suffers or is prone to a condition that can be prevented or treated by administering a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals and other non-mammalian animals.

"Therapeutically effective amount" refers to an amount of a conjugated biologically active agent or a pharmaceutical composition useful for treating, improving or preventing an identified disease or condition, or for presenting a detectable therapeutic or inhibitory effect. The effect can be detected by any test method known in the art.

The "biological half-life" of a substance is a pharmacokinetic parameter that specifies the time required for half of the substance to be removed from an organism after the introduction of the substance into the organism.

B. Compounds of the invention

Compounds of formula (la) are described herein:

(X- (Sp1) n) a-L- (K) b (Ia)

where

K is a group of the formula

image21

each occurrence of Q is independently selected from the group consisting of H and C1-4 alkyl;

each occurrence of T is independently selected from the group consisting of H, -C 1-4 alkyl, and -C 1-4 alkyl-O-

PC, where PC represents a phosphorylcholine group, with the proviso that one or more T groups are -C 1-4 alkyl-O-

PC;

each occurrence of E is independently selected from the group consisting of halo and Nu, in which Nu represents a nucleophilic group;

Sp1 is a spacer group;

X is a reactive group or a protected form thereof; n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000; a is an integer that varies from 1-8; b is an integer that varies from 1-8;

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L is selected from the group consisting of C 1-4 alkyl, cycloalkyl, carboxy-C 1-4 alkyl, carboxycycloalkyl, C 1-4 alkoxyC 1-4 alkyl and cyclic alkyl ether; and * indicates the point of attachment of each group K to said L.

In an embodiment of compounds of formula (Ia) K is a group of the formula

image22

each occurrence of Q is independently selected from the group consisting of H, methyl, and ethyl; each occurrence of T is independently selected from the group consisting of H, -CH3, -CH2-CH3, and -CH2-CH2-O-PC, where PC represents a phosphorylcholine group, with the proviso that one or more T groups are -CH2-CH2-O-PC; each occurrence of E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), - N (C1-4 alkyl) 2, -OH, -O- (C1- alkyl 4), and -O- (C1-4 haloalkyl);

Sp1 is selected from the group consisting of: -C1-12 alkyl-, -C3-12 -cycloalkyl-, - (C3-12 alkyl-C3-12 alkyl) - (C0-8 alkyl) -, - (CH2K12O-, (- ( CH2) 1-6-O- (CH2) 1-6-) 1-12-, (- (CH2) 1-4-NH- (CH2) 1-12-, (- (CH2) 1-4-O - (CH2) 1-4) 1-12-O-, KC ^^ - O ^ C ^ K

4-) 1-12O- (CH2) 1-12-, - (CH2) 1-12- (C = O) -O-, - (CH2) 1-12-O- (C = O) -, - (phenyl) - (CH2) 1-3- (C = O) -O-, - (phenyl) - (CH2) 1-3- (C = O) -NH-, - (C1-6 alkyl) - ( C = O) -O- (C0-6 alkyl) -, - (CH2) 1-12- (C = O) -O- (CH2) 1-12-, -CH (OH) -CH (OH) - (C = O) -O- -CH (OH) -CH (OH) - (C = O) -NH-, -S-maleimido- (CH2) 1-6-, -S-maleimido- (C1-alkyl) 3) - (C = O) -NH-, -S-maleimido- (C1-3 alkyl) - (C5-6 alkyl) - (C0-3 alkyl) -, - (C1-3 alkyl) - (Cs alkyl) -6) - (C0-3 alkyl) - (C = O) -O-, - (C1-3 alkyl) - (C5-cycloalkyl) - (C0-3 alkyl) - (C = o) - NH- , -S-maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) -, - (C0-3 alkyl) -phenyl- (C = O) -NH-, - (CH2) 1-12- NH- (C = O) -, - (^ 2) 1-12- (C = O) -NH-, - (phenyl) - (CH2) 1-3- (C = O) -NH-, -S - (CH2) - (C = O) -NH- (phenyl) -, - (CH2) 1-12- (C = O) -NH- (CH2) 1-12-, - (CH2) 2- (C = O) -O- (CH2) 2-O- (C = O) - (CH2) 2- (C = O) -NH-, - (C1-a alkyl) - (C = O) -N- ( C1-6 alkyl) -, acetal, ketal, acyloxyalkyl ether, -N = CH-, - (C1-6 alkyl) -SS- (C0-6 alkyl) -, - (C1-6 alkyl) -SS- (alkyl C1-6) - (C = O) -O-, - (C1-a-alkyl) -SS- (C1-a-alkyl) - (C = O) -NH-, -S-

5- (CH2) 1-3- (C = O) -NH- (CH2) 1-4-NH- (C = O) - (CH2) 1-3-, -SS- (C0-3 alkyl) - (phenyl) -, -SS- (C1-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) 1-5-, - (C1-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) 1-5- (C = O) -NH-, -SS- (C ^ alkyl) -, - (C1-3 alkyl) - (phenyl) - (C = O) -NH -, -O- (C1-C6 alkyl) -S (O2) - (C1-6 alkyl) -O- (C = O) -NH-, -SS- (CH2) 1-3- (C = O) -, - (CH2) 1-3- (C = O) -NH-N = CSS- (CH2) 1-3- (C = O) - NH- (CH2) 1-5-, - (CH2) 1 -3- (C = O) -NH- (CH2) 1-5- (C = O) -NH-, - (CH2) 0-3- (heteroaryl) - (CH2) 0-3-, - (CH2 ) 0-3-phenyl- (CH2) 0-3-,

, R2

R2

- N ^ C— - (aldui1 ^ l-6) -C — N- (Cj.g alkyl) -

R2 R3

\ ‘_

- (C- | .fi alkyl) - (ar¡l) -C = N (Cj alkyl ^) - - (Cj.6 alkyl) -C = N- (aryl) - (C j_6 alkyl) -

OR

C ^ alkyl - 0-P — O— (Co_g alkyl) OH

Y

X is selected from the group consisting of: hydroxyl, thiol, disulfide, dithiopyridyl, aldehyde, aldehyde hydrate, ketone, thione, acetal, ketal, -CH (OR1) 2, hemiacetal, hemicetal, monothiocetal, dithiohemicetal, dithiocetal, epoxide, thioepioxide , glyoxal, diones, amide, hydrazide, carboxyl, carboxylic acid ester, orthoester, N-hydroxysuccinimide ester, succinimidyl, maleimidyl, 1-benzotriazolyl, -CO2-succinimidyl, -CO2-maleimidyl, imido-ester, guanido, -CO2- (1- benzotriazolyl) amine, urea, carbamate, carbonate, thiourea, thiocarbamate, isocyanate, isothiocyanate, sulfone, chloroethylsulfone, substituted alpha-beta carbonyl, substituted alpha-beta carboxyl, acryloyl, acrylate, methacrylate, acrylamide, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone, vinylsulfone O (C = O) -CH2-I, -O (C = O) -CH2-Br, -NH (C = O) -CH2-I, -NH (C = O) -CH2-Br, - (C = O) -CH2-I, and - (C = O) -CH2-Br;

n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000; a is an integer that varies from 1-8; b is an integer that varies from 1-8;

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L is selected from the group consisting of C 1-4 alkyl, cycloalkyl, carboxy-C 1-4 alkyl, carboxycycloalkyl, C 1-4 alkoxyC 1-4 alkyl and cyclic alkyl ether;

R1 is C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms;

R2 is H, C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms; and * indicates the point of attachment of each group K to said L.

An embodiment of the present invention relates to compounds of formula (Ib):

(A-Z- (Sp1) n) a-L- (K) b (Ib)

where

K is a group of the formula

image23

in which the polymeric portion:

image24

of the compound is poly [2- (methacryloxyethyl) -2 '- (trimethylammonium) ethyl phosphate];

each occurrence of E is independently selected from the group consisting of halo and Nu, in which Nu represents a nucleophilic group;

Sp1 is a spacer group;

n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000; a is an integer that varies from 1-8; b is an integer that varies from 1-8;

L is selected from the group consisting of C1-4 alkyl, cycloalkyl, carboxy-C1-4 alkyl, carboxycycloalkyl, C1-4 alkoxy-C1-4 alkyl and cyclic alkyl ether;

A is a biologically active agent;

Z is the product of the reaction between a group present in said biologically active agent and a reactive group attached to said Sp1 group when n is 1, or a reactive group attached to L when n is 0; and * indicates the point of attachment of each group K to said L.

In an embodiment of compounds of formula (Ib), K is a group of the formula

image25

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twenty

25

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35

40

in which the polymeric portion:

image26

of the compound is poly [2- (methacryloxyethyl) -2 '- (trimethylammonium) ethyl phosphate];

each occurrence of E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), - N (C1-4 alkyl) 2, -OH, -O- (C1- alkyl 4), and -O- (C1.4 haloalkyl);

Sp1 is selected from the group consisting of: -C12-12 alkyl-, -C3-12 alkylcyclo-, - (C1-8 alkyl) - (C3-12 cycloalkyl) - (C0-8 alkyl) -, - (CH2) 1-12O-, (- (CH2) 1-6-O- (CH2) 1-6-) 1-12-, (- (C ^^ - NH ^ C ^) ^ - ^, (- (C ^ ^ -O ^ C ^^ - Om ^ O-, (- (CH2) 1-4-O-

(CH2) 1-4-) 1-12O- (CH2) 1-12-, - (CH2) 1-12- (C = O) -O-, - (CH2) 1-12-O- (C = O) -, - (phenyl) - (CH2) 1-3- (C = O) -O-, - (phenyl) - (CH2) 1-3- (C = O) - NH-, - (C1 alkyl -6) - (C = O) -O- (C0-6 alkyl) -, - (CH2) 1-12- (C = O) -O- (CH2) 1-12-, -CH (OH) - CH (OH) - (C = O) -O- -CH (OH) - CH (OH) - (C = O) -NH-, -S-maleimido- (CH2) 1-6-, -S-maleimido - (C1-3 alkyl) - (C = O) -NH-, -S-maleimido- (C1-3 alkyl) - (C5-e cycloalkyl) - (C0-3 alkyl) -, - (C1-3 alkyl ) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -O-, - (C1-3 alkyl) - (Cs-e cycloalkyl) - (C0-3 alkyl) - (C = O) -NH-, -S-maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) -, - (C0-3 alkyl) -phenyl- (C = O) -NH-, - (CH2 ) 1-12-NH- (C = O) -, - (CH2) 1-12- (C = O) -NH-, - (phenyl) - (CH2) 1-3- (C = O) -NH -, -S- (CH2) - (C = O) -NH- (phenyl) -, - (CH2) 1-12- (C = O) -NH- (CH2) 1-12-, - (CH2) 2- (C = O) -O- (CH2) 2-O- (C = O) - (CH2) 2- (C = O) -NH-, - (C1-e alkyl) - (C = O) -N- (C1-6 alkyl) -, acetal, ketal, acyloxyalkyl ether, - N = CH-, - (C1-6 alkyl) -SS- (C0-6 alkyl) -, - (C1-6 alkyl) - SS- (C1-6 alkyl) - (C = O) -O-, - (C1-6 alkyl) -SS- (C1-6 alkyl) - (C = O) - NH-, -SS- (CH2) 1-3- (C = O) -NH- (CH2) 1-4-NH- (C = O) - (CH2) 1-3-, -SS- (C0-3 alkyl) - (phenyl) -, -SS- (C1-3 alkyl) - (phenyl) - (C = O) - NH- ( CH2) 1-5-, - (C1-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) 1-5- (C = O) -NH-, -SS- (C ^ alkyl ) -, - (C1-3 alkyl) - (phenyl) - (C = O) -NH-, - O- (C1-C6 alkyl) -S (O2) - (C1-6 alkyl) -O- (C = O) -NH-, -SS- (CH2) 1-3- (C = O) -, - (CH2) 1-3- (C = O) -NH-N = CSS- (CH2) 1-3 - (C = O) - NH- (CH2) 1-5-, - (CH2) 1-3- (C = O) -NH- (CH2) 1-5- (C = O) -NH-, - (CH2) 0-3- (heteroaryl) - (CH2) 0-3-, - (CH2) 0-3-phenyl- (CH2) 0-3-,

image27

R2 R2

\ '

- (C- | .fi alkyl) - (arl) -C = N (Cj alkyl ^) - - (Cj alkyl.6) -C = N- (aryl) - (C! _ * alkyl) -

OR

Ci_j alkyl) ~ 0 — P — O- (alkyl Co. ^) -

OH;

Y

n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000; a is an integer that varies from 1-8; b is an integer that varies from 1-8;

L is selected from the group consisting of C1-4 alkyl, cycloalkyl, carboxy-C1-4 alkyl, carboxycycloalkyl, C1-4 alkoxy-C1-4 alkyl and cyclic alkyl ether;

A is a biologically active agent;

Z is selected from the group consisting of: phosphate, phosphate ester, - (C = O) O-, carboxylic acid ester, thioester, amide, disulfide, amine, -NH (R1) -, amidine, hydrazone, -N = CH-, -NH-CH2-,

^ R1

-N = C-

Y

-NH-C (R1) H-, -S-CH2-C (OH) (R2) -, -O-CH2-C (OH) (R2) -, -C (= O) O-CH2-C ( OH) (R2) -, -NR2-CH2-C (OH) (R2) -, -S-CH2- C (SH) (R2) -, -O-CH2-C (SH) (R2) -, - C (= O) O-CH2-C (SH) (R2) -, -NR2-CH2-C (SH) (R2) -,

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40

^ R2

-N = C-

-C (R2) H-NH-, - (C = O) -NH- (C = O) -NH-,

R2

-C = N- (C = 0) -N H

-C (R2) H-NH- (C = O) -NH-, - (C = O) -NH- (C = O) -O-,

R2

-C = N- (C = 0) -0-,

-C (R2) H-NH- (C = O) -O-, - (C = O) -NH- (C = S) -NH-,

R2

-C = N- (C = S) -NH

-C (R2) H-NH- (C = S) -NH-, - (C = O) -NH- (C = S) -O-,

R;

-C = N- (C = S) -0-

-C (R2) H-NH- (C = S) -O-,

C = N-NH

-NH- (C = O) -NH-, -O- (C = O) -NH-, -NH- (C = S) -NH-, -O- (C = S) -NH-, -S -CH2CH2- (C = O) -, -S-CH2CH (CH3) - (C = O) - S- CH2CH2- (C = O) O-, -S-CH2CH (CH3) - (C = O) O-, -S-CH2CH2- (C = O) NH-, -S-CH2-CH2- (pyridyl) -, -S-CH2-CH2-SO2-, -S-CH2- CH2-SO2-, -S -CH2- (C = O) -O-, -S-CH2- (C = O) -NH-, -S-CH2- (C = O) -, and

image28

R1 is C1.6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms;

R2 is H, C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms; Y

* indicates the point of attachment of each group K to said L.

B.1 Linear Compounds

Compounds of formula (la) or formula (Ib) in which a and b are both 1 (for example, a salt, hydrate or isomer thereof) are described herein. Such embodiments include, for example, compounds of formula (IIa) and compounds of formula (IIb), respectively.

Compounds of formula (IIa), or a salt, hydrate, or isomer thereof are described herein.

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image29

where

each occurrence of Q is independently selected from the group consisting of H, methyl, and ethyl; each occurrence of T is independently selected from the group consisting of H, -CH3, -CH2-CH3, and -CH2-CH2-O-PC, where PC represents a phosphorylcholine group, with the proviso that one or more T groups are -CH2-CH2-O-PC; E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), -N (^ -4 alkyl) 2, - OH, -O- (C1-4 alkyl), and -O- (C1-4 alkyl substituted with fluorine);

Sp1 is a spacer group;

X is a reactive group or a protected form thereof; n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000; Y

L is selected from the group consisting of C1-4 alkyl, cycloalkyl, carboxy-C1-4 alkyl, carboxycycloalkyl, C1-4 alkoxy-C1-4 alkyl and cyclic alkyl ether.

A zwitterionic compound of formula (IIb) or a salt, hydrate, or isomer thereof is also described herein:

image30

where

each occurrence of Q is independently selected from the group consisting of H, methyl, and ethyl; each occurrence of T is independently selected from the group consisting of H, -CH3, -CH2-CH3, and -CH2-CH2-O-PC, where PC represents a phosphorylcholine group, with the proviso that one or more T groups are -CH2-CH2-O-PC; E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), -N (C1-4 alkyl) 2, - OH, -O- (C1-4 alkyl), and -O- (C1.4 alkyl substituted with fluorine);

Sp1 is a spacer group;

n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000;

L is selected from the group consisting of C1-4 alkyl, cycloalkyl, carboxy-C1-4 alkyl, carboxycycloalkyl, C1-4 alkoxy-C1-4 alkyl and cyclic alkyl ether;

A is a biologically active agent; Y

Z is the product of the reaction between a group present in said biologically active agent and a reactive group attached to said Sp1 group when n is 1, or a reactive group attached to L when n is 0.

Unless otherwise specified, all variable groups present in the compounds of the formulas (IIa) and (IIb) are as defined for the compounds of the formulas (Ia) and (Ib) and embodiments thereof, respectively .

B.2 Nonlinear compounds

The present invention relates to compounds that have more complex architectures. Such architectures include branched compounds, bifurcated compounds, branched and bifurcated compounds. Branched polymeric compounds 5 having multiple polymer arms can also have comb or star architectures. In theory, branched compounds that have two or more polymer arms can help promote water solubility without unduly hampering the interactions of a bound biologically active agent.

One aspect of the present invention is directed to bifurcated compounds of formula (Ia) or formula (Ib), where a is an integer greater than 1. In one embodiment, the bifurcated compounds of formula (Ia) or formula (Ib) they can have a = 2 and b = 1, for example, as in the compounds of formula (IIIa) (i.e. (X- (Sp1) n) 2-L- (K) -i) or formula (IIIb) (is say, (AZ- (Sp1) n) 2-L- (K) 1).

image31

fifteen

in which each polymeric portion:

image32

20 of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl phosphate].

In other embodiments, the bifurcated compounds of formula (Ia) or formula (Ib) a = 3 and b = 1; and in other embodiments a = 4 and b = 1.

In other embodiments of compounds of formula (la) or formula (Ib), b is an integer greater than 1 and the compounds are branched compounds having more than one polymeric arm in the form of group K. In some embodiments of branched compounds of formula (Ia) or formula (Ib), a = 1 and b = 2, as in, for example, the compounds of formula (IVa) (i.e. (X- (Sp1) n) 1-L- (K) 2) or formula (IVb) (i.e. (AZ- (Sp1) n) 1-L- (K) 2).

5

10

image33

image34

of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate.

In other embodiments of branched compounds of formula (Ia) or formula (Ib), a = 1 and b = 3; and in yet another embodiment a = 1 and b = 4. In those embodiments in which b is an integer greater than 1, (for example, b is 2, 3 or 4), the polymeric portion of the molecule may be poly- hydroxyethylmethacryloyl phosphorylcholine, regardless of the value of a (for example, a may be 1, 2, 3 or 4).

In other embodiments, more than compounds of formula (la) or formula (Ib), both a and b are whole numbers greater than 1 and the compounds are branched and branched compounds. In one embodiment of branched and bifurcated compounds of formula (Ia) or formula (Ib), a = 2 and b = 2, for example, as in compounds of formula (Va) (i.e. (X- (Sp1) n) 2-L- (K) 2) and the compounds of formula (Vb) (ie (AZ- (Sp1) n) 2-L- (K) 2).

image35

5

image36

of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate.

In other embodiments of branched and bifurcated compounds of formula (Ia) or formula (Ib), a = 2 and b = 3 or 4. In

10 other embodiments of branched and bifurcated compounds of formula (Ia) or formula (Ib), a = 3 or 4 and b = 2.

In some embodiments of branched, bifurcated, or branched and bifurcated compounds, where the sum of a and b is 3; L comprises a carbon atom; or in another embodiment, 2 carbon atoms, or in another embodiment, L comprises 3 or more carbon atoms. In other embodiments of branched, bifurcated, or branched and bifurcated compounds, where the sum of a and b is 4; L comprises 2 carbon atoms, or in another embodiment 3

carbon atoms, or in another embodiment 4 carbon atoms. In still other embodiments of compounds

branched, bifurcated, or branched and bifurcated, where the sum of a and b is 5; L comprises 3 carbon atoms, or in another embodiment 4 carbon atoms, or in another embodiment 5 carbon atoms. Still in others

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Four. Five

fifty

embodiments of branched, bifurcated, or branched and bifurcated compounds, where the sum of a and b is 6; L comprises 4 carbon atoms, or in another embodiment 5 carbon atoms, or in another embodiment 6 carbon atoms.

Unless otherwise specified, all variable groups present in the compounds of formulas (IIIa), (IVa) or (Va) are as defined for the compounds of formula (Ia) or embodiments thereof. Similarly, unless otherwise specified, all groups present in the compounds of the formulas (IIIb), (IVb) or (Vb) are as defined for the compounds of formula (Ib) or embodiments of the same.

B.3 Compounds of formulas (Ia) to (Vb)

In compounds of the formulas (Ia) to (Vb) (i.e., the compounds (Ia), (Ib), (IIa), (IIb), (IIIa), (IIIb), (IVa), (IVb), (Va), or (Vb)), the polymeric portion of the compounds comprises poly (2-methacryloxyethyl phosphorylcholine) also referred to as pMPC, poly-hydroxyethylmethacryloyl phosphorylcholine, pHEMA-PC or poly-HEMA-PC. In other embodiments of compounds of the formulas (Ia) to (Vb), the polymeric portion of the molecule consists of a poly-HEMA-PC polymer, for example, K is a group of the formula

image37

where Q is methyl; T is a phosphorylcholine group; E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), -N (C1-4 alkyl) 2, -OH, -O- (C1-4 alkyl), and -O- (C1-4 alkyl substituted with fluorine); and m is an integer selected based on the desired molecular weight of the polymeric portion of a compound of formulas (Ia) a (Vb).

In some embodiments of compounds of the formulas (Ia) (IIa), (IIIa), (IVa), or (Va), m is an integer ranging from about 2 to about 30, or from about 30 to about 100, or from about 20 to about 200, or alternatively, from about 100 to about 500. In another embodiment, m is an integer ranging from about 500 to about 1,000; and in yet another embodiment, m is an integer that ranges from about 1,000 to about 2,000. The compounds of the formulas (Ia) (IIa), (IIIa), (IVa), or (Va) can also have the value of m adjusted such that the molecular weight range (in Daltons) of the compound is approximately 500 to about 2000, or 2,000 to about 5,000, or about 5,000 to about 10,000, or about 10,000 to about 50,000, or about 50,000 to about 100,000, or about 100,000 to about 200,000. Exemplary molecular weights for compounds of approximately approximately approximately approximately approximately approximately approximately approximately approximately approximately

approximately 150,000 Daltons and approximately 175,000 Daltons.

 the formulas (Ia)

 (IIa), (IIIa), (IVa), or (Va) include approximately 2,000 Daltons

 2,200

 Daltons, approximately 2,500 Daltons, approximately 3,000 Daltons,

 4,000

 Daltons, approximately 4,400 Daltons, approximately 5,000 Daltons,

 6,000

 Daltons, approximately 7,000 Daltons, approximately 7,500 Daltons,

 8,000

 Daltons, approximately 9,000 Daltons, approximately 10,000 Daltons,

 11,000

 Daltons, approximately 12,000 Daltons, approximately 13,000 Daltons

 14,000

 Daltons, approximately 15,000 Daltons, approximately 20,000 Daltons

 22,500

 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons

 40,000

 Daltons, approximately 50,000 Daltons, approximately 60,000 Daltons

 75,000

 Daltons, approximately 80,000 Daltons, approximately 90,000 Daltons

 100,000

 Daltons, approximately 120,000 Daltons, approximately 140,000 Daltons,

In some embodiments of compounds of the formulas (Ib), (IIb), (IIIb), (IVb), or (Vb) m is an integer ranging from about 2 to about 30, or from about 30 to about 100, or from about 20 to about 200, or alternatively, from about 100 to about 500. In some embodiments, m is an integer that ranges from about 500 to about

1,000, and in other embodiments, m is an integer that ranges from about 1,000 to about

2,000 The compounds of the formulas (Ib), (IIb), (IIIb), (IVb), or (Vb) can also have the value of m adjusted such that the molecular weight range (in Daltons), excluding weight molecular agent biologically

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40

Four. Five

fifty

55

60

65

asset A, is from about 2,000 to about 5,000, or from about 5,000 to about 10,000, or from about 10,000 to about 50,000, or from about

50,000 to about 100,000, or from about 100,000 to about 200,000. Exemplary molecular weights for the compounds of the formulas (Ib) (IIb), (IIIb), (IVb), or (Vb) excluding the molecular weight of the biologically active agent A, include approximately 2,000 Daltons, approximately 2,200 Daltons, approximately 2,500 Daltons, approximately 3,000 Daltons, approximately 4,000 Daltons, approximately approximately approximately approximately approximately approximately approximately approximately 175,000 Daltons.

 4,400

 Daltons, approximately 5,000 Daltons, approximately 6,000 Daltons,

 7,000

 Daltons, approximately 7,500 Daltons, approximately 8,000 Daltons,

 9,000

 Daltons, approximately 10,000 Daltons, approximately 11,000 Daltons,

 12,000

 Daltons, approximately 13,000 Daltons, approximately 14,000 Daltons,

 15,000

 Daltons, approximately 20,000 Daltons, approximately 22,500 Daltons,

 25,000

 Daltons, approximately 30,000 Daltons, approximately 40,000 Daltons,

 50,000

 Daltons, approximately 60,000 Daltons, approximately 75,000 Daltons,

 80,000

 Daltons, approximately 90,000 Daltons, approximately 100,000 Daltons,

 120,000

 Daltons, approximately 140,000 Daltons, approximately 150,000 Daltons and

In some embodiments of compounds of the formulas (Ia) or (Ib), which have multiple polymer arms where b is greater than or equal to 3, the value of m can be selected such that the molecular weight of the compounds, excluding The weight of the active agent "A", when present, is from about 2,000 to about

300,000 Daltons. Other molecular weight ranges for the compounds of formula (Ia) or (Ib) where b is greater than or equal to 3 are from about 5,000 to about 10,000, or from about 10,000 to about 50,000, or from about 50,000 to about 100,000, or about

100,000 to about 200,000 or from about 200,000 to about 250,000 or from about 250,000 to about 300,000 Daltons, excluding the weight of active agent "A" when present.

The length of the polymer in compounds of formula (Ia) to (Vb), and therefore the mass, can be varied above at least the ranges indicated above. Depending on the synthetic media employed and the subsequent purification methods employed, the variation in the size / mass of the compounds of formula (Ia) to (Vb) Ib can be controlled to give preparations having narrowly dispersed intervals, or alternatively, intervals of widely dispersed size / mass.

In some embodiments of compounds of the formulas (Ia), (IIa), (IIIa), (IVa), or (Va), X is a reactive group or a protected form thereof that can react with nucleophiles or electrophiles. In some embodiments, X is a group capable of reacting with a nucleophile or electrophile present in a biologically active agent. In other embodiments, X is a group capable of reacting with a functionality selected from an aldehyde, a ketone, an amine, a carboxyl, a hydroxyl, a guanido or thiol group of a biologically active agent to form a covalent bond between the biologically active agent. active and the group L to which X was attached. The binding between the biologically active agent and L may include one or more atoms derived from X, one or more atoms derived from the reaction of A with X, or atoms of a group Sp1, if present Alternatively, the union between L and the biologically active agent can be a direct covalent bond where X is a group that can be displaced from L, such as by a nucleophile of the biologically active agent acting in a nucleophilic substitution reaction SN1 or SN2.

In other embodiments of compounds of the formulas (Ia) (IIa), (IIIa), (IVa), or (Va) X is selected from the group consisting of: hydroxyl, thiol, disulfide, dithiopyridyl, aldehyde, aldehyde hydrate, ketone , tiona, acetal, ketal, -CH (OR1) 2, hemiacetal, hemicetal, monothiocetal, dithiohemicetal, dithiocetal, epoxide, thioepoxide, glyoxal, diones, amide, hydrazide, carboxyl, carboxylic acid ester, orthoester, N-hydroxysuccinimide ester succinimidyl, maleimidyl, 1- benzotriazolyl, -CO2-succinimidyl, -CO2-maleimidyl, imido-ester, guanido, -CO2- (1-benzotriazolyl) amine, urea, carbamate, carbonate, thiourea, thiocarbamate, isocyanate, isothiocyanate, sulfonacyanate chloroethylsulfone, substituted alpha-beta carbonyl, substituted alpha-beta carboxyl, acryloyl, acrylate, methacrylate, acrylamide, vinylsulfone, vinyl pyridine, -O (C = O) - CH2-I, -O (C = O) -CH2-Br, -NH (C = O) -CH2-I, -NH (C = O) -CH2-Br, - (C = O) -CH2-I, and - (C = O) -CH2-Br; wherein R1 is C1-6 alkyl, C3-C6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms.

In some embodiments of compounds of the formulas (Ia), (IIa), (IIIa), (IVa), or (Va), X is a group selected from protected and unprotected forms of: hydroxyl, thiol, disulfide, dithiopyridyl, aldehyde, aldehyde hydrate, ketone, tione, acetal, ketal, -CH (OR1) 2, hemiacetal, hemicetal, monothiocetal, dithiohemicetal, dithiocetal, epoxide, glyoxal, dionas, amide, hydrazide, carboxyl, carboxylic acid ester, orthoester , maleimidyl, 1-benzotriazolyl, -CO2-succinimidyl, -CO2-maleimidyl, and -CO2- (1-benzotriazolyl); wherein R1 is C1-C6 alkyl, C3-C6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms.

In other embodiments of compounds of the formulas (Ia) (IIa), (IIIa), (IVa), or (Va), X is a group selected from protected and unprotected forms of: ether, thioether, amine, urea, carbamate , carbonate, thiourea, thiocarbamate, isocyanate, isothiocyanate, sulfone, and chloroethylsulfone.

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55

In still other embodiments of compounds of the formulas (la) (Ila), (Illa), (IVa), or (Va), X is substituted alpha-beta carbonyl, C1-C12 alkenyl, acryloyl, acrylate, methacrylate, acrylamide, vinyl sulfone and vinyl pyridine. When a group X contains a double bond that cannot be present in the polymerization reactions, such as when X comprises a vinyl pyridine group, an unsaturated alpha-beta carbonyl, an alpha-beta ester, vinyl sulfone or an alpha-beta amide, The group can be introduced into the compounds after any polymerization process that creates the polymeric portion of the compounds of the invention. This can be achieved, for example, through a bifunctional agent having an X group and a group capable of reacting with a functionality linked to the L group, such as an NHS ester group, both added to a group - (Sp1) -.

Within the compounds of formula (Ia) a (Vb), when n is 0, the spacer group Sp1 is not present and -Sp1- represents a covalent bond such that L is directly linked to -ZA or -X by a covalent bond.

When n is 1, -Sp1- is present between residues A-Z and L or between residues X and L of compounds having formulas (Ia) to (Vb). In some embodiments, Sp1 is selected from the group consisting of: an alkyl ether and alkyl ester of an alkyl carboxylic acid. In other embodiments, Sp1 is selected from the group consisting of: -C12-12 alkyl-, -C3-12 alkyl-, - (C1-8 alkyl) - (C3-12 cycloalkyl) - (C0-8 alkyl) -, - (CH2) 1-12O-, (- (CH2) 1-6-O- (CH2) 1-e-) 1-12-, (-

(CH2) 1-4-NH- (CH2) 1-4) 1-12-, (- (CH2) 1-4-O- (CH2) 1-4) 1-12-O-, (- (CH2 ) 1-4-O- (CH2) 1-4-) 1-12O- (CH2) 1-12-, - (CH2) 1-12- (C = O) -O-, - (CH2) 1- 12-O- (C = O) -, - (phenyl) - (CH2) 1-3- (C = O) -O-, - (phenyl) - (CH2) 1-3- (C = O) - NH-, - (C1-6 alkyl) - (C = O) -O- (C0-6 alkyl) -, - (CH2) 1-12- (C = O) -O- (CH2) 1-12- , -CH (OH) -CH (OH) - (C = O) -O - CH (OH) -CH (OH) - (C = O) -NH-, -S-maleimido- (CH2) 1- 6-, -S- maleimido- (C1-3 alkyl) - (C = O) -NH-, -S-maleimido- (C1-3 alkyl) - (Cs-e cycloalkyl) - (C0-3 alkyl) - , - (C1-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -O-, - (C1-3 alkyl) - (Cs-e cycloalkyl) - (C0 alkyl -3) - (C = O) -NH-, -S-maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) -, - (C0-3 alkyl) -phenyl- (C = O) -NH-, - (CH2) 1-12-NH- (C = O) -, - (CH2) 1-12- (C = O) -NH-, - (phenyl) - (CH2) 1-3- (C = O) -NH-, - S- (CH2) - (C = O) -NH- (phenyl) -, - (CH2) 1-12- (C = O) -NH- (CH2) 1- 12-, - (CH2) 2- (C = O) -O- (CH2) 2-O- (C = O) - (CH2) 2- (C = O) -NH-, -

(C1-6 alkyl) - (C = O) -N- (C1-6 alkyl) -, acetal, ketal, acyloxyalkyl ether, -N = CH-, - (C1-a alkyl) -SS- (C0- alkyl 6) -, - (C1-6 alkyl) -SS- (C1-6 alkyl) - (C = O) -O-, - (C1-a alkyl) -SS- (C1-6 alkyl) - (C = O) -NH-, -SS- (CH2) 1-3- (C = O) -NH- (CH2> m-NH- (C = O) - (CH2) 1-3-, -SS- (alkyl C0-3) - (phenyl) -, -SS- (C1-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) 1-5-, - (C1-3 alkyl) - (phenyl ) - (C = O) -NH- (CH2) 1- 5- (C = O) -NH-, -SS- (C1.3 alkyl) -, - (C1-3 alkyl) - (phenyl) - ( C = O) -NH-, -O- (C1-C6 alkyl) -S (O2) - (C1-6 alkyl) -O- (C = O) -NH-, -S- S- (CH2) 1 -3- (C = O) -, - (CH2) 1-3- (C = O) -NH-N = CSS- (CH2) 1-3- (C = O) -NH- (CH2) 1- 5-, - (CH2) 1-3- (C = O) -NH- (CH2) 1-5- (C = O) - NH-, - (CH2) 0-3- (heteroaryl) - (CH2) 0-3-, - (CH2) 0-3-phenyl- (CH2) 0-3-,

image38

R2 R3

\ '

- (C | .íi alkyl) - (ar¡l) -C = N (C ^ alkyl) - - (Cj.6 alkyl) -C = N- (aryl) - (C j_6 alkyl) -

OR

alkyl Ci _ ^) ~ 0 — P — O— (alkyl Co_g)

OH

Y

In another embodiment, Sp1 is selected from the group consisting of: -C1-C12-alkyl, -C3-C12-alkyl-alkyl, (- (CH2) 1-6-O- (CH2) 1-6-) 1-12- , (- (CH2) 1-4-NH- (CH2) 1-4) 1-12-, - (CH2) 1-12O-, (- (CH2) 1-4-O- (CH2) 1-4 ) 1-12-O-, - (CH2) 1-12- (CO) -O-, - (CH2) 1-12- (CO) -NH-, - (CH2) 1-12-O- (CO ) -, - (CH2) 1-12-NH- (CO) -, (- (CH2) 1-4-O- (CH2) 1-4) 1-12-O- (CH2) 1-12-, - (CH2) 1-12- (CO) -O- (CH2) 1-12-, - (CH2) 1-12- (CO) -NH- (CH2) 1-12-, - (CH2) 1- 12-O- (CO) - (CH2) 1-12-, - (CH2) 1-12-NH- (CO) - (CH2) 1-12-, - (C3-C12 cycloalkyl) -, - (alkyl C1-C8) - (C3-C12 cycloalkyl) -, - (C3-C12 cycloalkyl) - (C1-8 alkyl) -, - (C1-8 alkyl) - (C3-C12 alkyl) - (C1-8 alkyl) - and - (CH2) 0-3-aryl- (CH2) 0-3-. When the Sp1 groups are present in a molecule of the formulas (Ia) to (Vb), the Sp1 group is preferably oriented such that a carbon atom of Sp1 is directly linked to X by a covalent bond.

Z is the product of the reaction between a group present in a biologically active agent and a reactive group (for example, a group X of a compound of the formulas (Ia) (IIa), (IIIa) (IVa) or (Va) ) attached to a Sp1 group when n is 1, or to L when n is 0. In some embodiments of compounds (Ib) (11 b), (IIIb), (IVb) or (Vb), Z is formed from a nucleophile or electrophile present in a biologically active agent and an X group that is capable of forming one or more covalent bonds through a reaction with the nucleophile or electrophile present in the biologically active agent. In other embodiments, Z is a group resulting from the formation of a covalent bond between an X group and an aldehyde, a ketone, a carboxyl group, an amine, a hydroxyl or a thiol present in a biologically active group.

Some non-limiting examples of Z groups formed from the reaction of some representative X groups and some groups typically found or introduced into biologically active agents are set forth in the

Table I.

Table I

 Biologically active agents and illustrative groups that can react with a reactive group (X) to form a Z group.

 Exemplary reactive X groups of the compounds (la), (Ila), (Illa), (IVa), or (Va) (shown attached to - (Sp1) n-J) Product A-Z- (Sp1) n-t

 A-COOH

 HO- (Sp '') n-t A-C (= O) O- (Sp1) n-t

 hydroxyl or activated forms thereof (eg, tresylate, mesylate, etc.)

 A-COOH A-SH

 HS- (SpVt thiol A-C (= O) S- (Sp1) n-í A-S-S- (Sp1) n-t

 A-SH

 R1-S-S- (Sp1) n-Í (disulfide) A-S-S- (Sp1) n-í

 A-SH

 (pyridyl) -S-S- (Sp1) n-í (dithiopyridyl) A-S-S- (Sp1) n-í

 A-NH2

 H (O =) C- (Sp ') n-t aldehyde A-N = CH- (Sp1) n-t or A-NH-CH2- (Sp1) n-t after reduction

 A-NH2

 (HO) 2HC- (Sp ') n-í aldehyde hydrate A-N = CH- (Sp1) n-t o

 A-NH-CH2- (Sp1) n-t after reduction

 A-NH2

 (R '0) 2CH- (Sp') n-t 0 r— ° v h / (Sp1) n "í 1 — O A-N = CH- (Sp1) n-t or A-NH-CH- (Sp1) n-f after reduction

 acetal

 A-NH2

 R'OCHfOHJ-fSp'jn-t or hemiacetal A-N = CH- (Sp1) n-t or A-NH-CH- (Sp1) n-f after reduction

 A-NH2

 R1 (O =) C- (Sp1) n-í ketone A-N = CR1- (Sp1) n-t or A-NH-C (R1) H- (Sp1) n-t after reduction

 A-NH2

 (R1O) 2CH- (Sp1) „- tor — 0 r1 (Sp1) n- + —0 ketal AN = C (R1) - (Sp1) nt or A-NH-C (R1) H- (Sp1) nt after The reduction

 A-NH2

 R1OCH (OH) - (Sp1) n-t hemicetal A-N = C (R1) - (Sp1) n-t or A-NH-C (R1) H- (Sp1) n-t after reduction

 Biologically active agents and illustrative groups that can react with a reactive group (X) to form a Z group.

 Exemplary reactive X groups of the compounds (la), (Ila), (IlIa), (IVa), or (Va) (shown attached to - (Sp1) n-J) Product A-Z- (Sp ') n-t

 A-NH2

 R '(S =) C- (Spl) n-í thione ketone (thioketone) A-N = C (R') - (Sp ') n-t or A-NH-C (R1) H- (Sp1) n-t after reduction

 A-NH2

 (R1O) (R1S) CH- (Sp1) n-t or A-N = C (R1) - (Sp1) n-t

 r-o r1 / ~ (Sp1) n * + 1 —- c or A-NH-C (R1) H- (Sp1) n-t after reduction

 monotiocetal

 A-NH2

 R1SCH (SH) - (Sp1) n-t or dithiohemicetal A-N = C (R1) - (Sp1) n-t or A-NH-C (R1) H- (Sp1) n-t after reduction

 A-NH2

 (R1S) 2CH- (Sp1) „- t or A-N = C (R1) - (Sp1) n-t

 r-s r1 / “(Sp1) n- + L ---- 0 or A-NH-C (R1) H- (Sp1) n-t after reduction

 dithiocetal

 A-SH

 R2 ^> L- (SP1) n * í A-S-CH2-C (OH) (R2) (Sp1) n-t

 A-OH A-COOH (anion) A-NHR2

 epoxy (oxirane) AO-CH2-C (OH) (R2) (Sp1) nt AC (= O) O-CH2-C (OH) (R2) (Sp1) n-Í A-NR2-CH2-C (OH ) (R2) (Sp1) nt

 A-SH A-OH A-COOH (anion) A-NHR2

 R2 (Sp1U thioepoxide AS-CH2-C (SH) (R2) - (Sp1) nt AO-CH2-C (SH) (R2) (Sp1) nt AC (= O) O-CH2-C (SH) (R2 ) (Sp1) n-Í A-NR2-CH2-C (SH) (R2) (Sp1) nt

 A-SH

 HO- (C = O) - (Sp1) n-í carboxyl A-S- (C = O) - (Sp1) n-í

 A-OH A-NHR2

   A-O- (C = O) - (Sp1) n-t A-NH- (C = O) - (Sp1) n-t

 A-sh A-oh A-NHr2

 (alcohol) - (C = O) - (Sp1) nf carboxylic acid ester (alcohol indicates a suitable esterified alcohol leaving group, for example, p-nitrophenyl) AS- (C = O) - (Sp1) n- í AO- (C = O) - (Sp1) nt A-NH- (C = O) - (Sp1) nt

 Biologically active agents and illustrative groups that can react with a reactive group (X) to form a Z group.

 Exemplary reactive X groups of the compounds (la), (Ila), (Illa), (IVa), or (Va) (shown attached to - (Sp1) n-J) Product A-Z- (Sp1) n-t

 A-NH2

 O A N-0-R3- (Spl) „t 0 N-hydroxysuccinimide ester A-NH-R3- (Sp1) n-í

 A-sh2

 0 ■ A N (Spl) n 0 0 A-S ^ \ N— (Spl) n-Í 0

 A-NH2

 £ / / l-R¿- (SplU "N 1-benzotriazol ester A-NH-R3- (Sp1) n-í

 A-NH2

 CH3- (CH2) i-3) -O (C = NH) - (S '') n-t (imido-ester) A-NH- (C = NH) - (Sp1) n-í (amidine)

 A- (C = NH) -O (CH2) i-3) -CH3 (imido ester)

 H2N- (Sp1) n-í A- (C = NH) -HN- (Sp1) n-í (amidine)

 A-COOH A- (C = O) -R2

 H2N- (Sp1) n-í amine A- (C = O) -NH- (Sp1) „- t A- (R2) C = N- (Sp1) n-í or A- (R2) CH- NH- (Sp1) nt after reduction

 A-COOH A- (C = O) -R2

 H2N- (C = O) -NH- (Sp1) nt urea A- (C = O) -NH- (C = O) -NH- (Sp1) nt A- (R2) C = N- (C = O ) -NH- (Sp1) nt or A- (R2) CH-NH- (C = O) -NH- (Sp1) n- t after reduction

 A-COOH A- (C = O) -R2

 H2N- (C = O) -O- (Sp'Vt carbamate A- (C = O) -NH- (C = O) -O- (Sp1) nt A- (R2) C = N- (C = O ) -O- (Sp1) nt or A- (R2) CH-NH- (C = O) -O- (Sp1) nt after reduction

 A-COOH A- (C = O) -R2

 H2N- (C = S) -NH- (Sp1) n-í thiourea A- (C = O) -NH- (C = S) -NH- (Sp1) nt A- (R2) C = N- (C = S) -NH- (Sp1) nt or A- (R2) CH-NH- (C = S) -NH- (Sp1) nt after reduction

 Biologically active agents and illustrative groups that can react with a reactive group (X) to form a Z group.

 Exemplary reactive X groups of the compounds (la), (Ila), (IlIa), (IVa), or (Va) (shown attached to - (Sp1) n-t) Product A-Z- (Sp1) n-t

 A-COOH A- (C = O) -R2

 H2N- (C = S) -O- (Spl) „- t thiocarbamate A- (C = O) -NH- (C = S) -O- (Sp1) nt A- (R2) C = N- (C = S) -O- (Sp1) nt or A- (R2) CH-NH- (C = S) -O- (Sp1) nt after reduction

 A- (C = O) -R2

 H2N-HN- (Sp1) n-t A- (R2) C = N-HN- (Sp1) n-t

 hydrazone

 A-NH-NH2

 R2- (O = C) - (Sp1) n-í A-NH-N = C (R2) - (Sp1) n-t hydrazone

 A-NH2

 O = C = N- (Sp1) n-t isocyanate A-NH- (C = O) -NH- (Sp1) n-t

 A-OH

   A-O- (C = O) -NH- (Sp1) n-t

 A-NH2

 S = C = N- (Sp1) n-í isothiocyanate A-NH- (C = S) -NH- (Sp1) n-t

 A-OH

   A-O- (C = S) -NH- (Sp1) n-t

 A-SH

 H2C = CH- (C = O) - (Sp1) n-t A-S-CH2CH2- (C = O) - (Sp1) n-í

 or H2C = C (CH3) - (C = O) - (Sp1) n-t alpha-beta carbonyls without replacing A-S-CH2-CH (CH3) - (C = O) - (Sp1) n- í

 A-SH

 H2C = CH- (C = O) O- (Sp1) n-t carboxyl alpha-beta without replacing A-S-CH2CH2- (C = O) O- (Sp1) n-t

 A-SH

 H2C = C (CH3) - (C = O) -O- (Sp1) n-í unsubstituted alpha-beta carboxyls (methacrylates) A-S-CH2CH (CH3) - (C = O) O- (Sp1) n-í

 A-SH

 H2C = CH- (C = O) NH- (Sp1) n-t unsubstituted alpha-beta amides (acrylamides) A-S-CH2CH2- (C = O) NH- (Sp1) n-í

 A-SH

 vinylpyridine- (Sp1) n-f (2- or 4-vinylpyridine) A-S-CH2-CH2- (pyridyl) - (Sp1) n-í

 A-SH

 H2C = CH-SO2- (Sp1) n-t (vinyl sulfone) A-S-H2C-CH2-SO2- (Sp1) n-í

 A-SH

 ClH2C-CH2-SO2- (Sp1) n (chloroethyl sulfone) A-S-H2C-CH2-SO2- (Sp1) n-í

 A-SH

 (halogen) -CH2- (C = O) -O- (Sp1) nt (halogen) -CH2- (C = O) -NH- (Sp1) nt (halogen) -CH2- (C = O) - (Sp1 ) nt (halogen is preferably I or Br) AS-CH2- (C = O) -O- (Sp1) n-í AS-CH2- (C = O) -NH- (Sp1) „- í AS-CH2- (C = O) - (Sp1) n-í

 A-O (C = O) -CH2- (halogen)

 HS-fSp'Vt A-O (C = O) -CH2-S- (Sp1) n-í A-NH (C = O) -CH2-S- (Sp1) n-í

 A-NH (C = O) -CH2- (halogen) A- (C = O) - CH2- (halogen) (halogen is preferably I or Br)

   A- (C = O) -CH2-S- (Sp1) n-í

 A-SH

 (halogen) -CH2 (C = O) O- (Sp1) nt (halogen) -CH2 (C = O) NH- (Sp1) nt AS-CH2 (C = O) O- (Sp1) n-í AS- CH2 (C = O) NH- (Sp1) n-í

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 Biologically active agents and illustrative groups that can react with a reactive group (X) to form a Z group.

 Exemplary reactive X groups of the compounds (Ia), (IIa), (IIIa), (IVa), or (Va) (shown attached to - (Sp1) n-t) Product A-Z- (Sp ') n-t

 (halogen) -CH2 (C = O) - (Sp1) n-t (halogen is preferably I or Br) A-S-CH2 (C = O) - (Spl) „- t

R1 is Ci-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms;

R2 is H, C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms;

R3 is a carbonyl derivative * - (C = O) -, * - (C = O) - (CH2) 1-8-SS-, * - (C = O) - (CH2) 1-8- (C = O) -O-, * - (C = O) - (CH2) 1-8-O- (C = O) -, * - (C = O) - (CH2) 1-8- (C = O) -NH-, or * - (C = O) - (CH2) 1-8-NH- (C = O) -, or alternatively, R3 is a carbonyl derivative of the form * - (C = O) -O - (CH2) 1-8-SS-, * - (C = O) -O- (CH2) 1-8- (C = O) -O-, * - (C = O) -O- (CH2) 1-8-O- (C = O) -, * - (C = O) -O- (CH2) 1-8- (C = O) -NH-, or * - (C = O) -O- (CH2) 1-8-Nh- (C = O) -, where "*" indicates the point of attachment to succinimidyl or benzotriazolyl groups;

N is independently 0 or 1; and $ indicates the point of attachment to a group L.

The biologically active agent, A, of compounds of formula (Ib), (IIb), (Illb), (IVb), or (Vb) may include a spacer group, (Sp2) p, as a means of binding the active agent , A, to Z. When p is 0, the spacer group Sp2 is not present and -Sp2- represents a covalent bond such that the biologically active agent "A" binds directly to the group Z via one or more covalent bonds ( for example, a double link). When n is 1, the spacer group represented Sp2 is present between groups A and Z, and the compounds of formula (Ib) can be written as (A (Sp2) pZ- (Sp1) n) aL- (K) b, where all components, except (Sp2) and p are as defined for the compounds of formula (Ib); and p is 0 or 1. In some embodiments, the Sp2 spacer groups may comprise an alkyl ether and an alkyl ester of an alkyl carboxylic acid.

In other embodiments of compounds of formula (Ib), (I Ib), (IIIb), (IVb), or (Vb), Sp2 is selected from the group consisting of: C1-12 alkyl, C3-12 alkylcyclo -, - (C1-8 alkyl) - (C3-12 cycloalkyl) - (C0-8 alkyl) -, - (CH2) 1-12O-, (- (CH2) 1-6-O- (CH2) 1- 6-) 1-12, (- (CH2) 1-4-NH- (CH2) 1-4) 1-12-, (- (CH2) 1-4-O- (CH2) 1-4) 1- 12-O-, (- (CH2) 1-4-O- (CH2) 1-4-) 1-12O- (CH2) 1-12-, - (CH2K 12- (C = O) -O-, - (CH2) 1-12-O- (C = O) -, - (phenyl) - (CH2) 1-3- (C = O) -O-, - (phenyl) - (CH2) 1-3- (C = O) -NH-, - (C1-6 alkyl) - (C = O) -O- (Cü-6 alkyl) -, - (CH2) 1-12- (C = O) -O- ( CH2) 1-12-, -CH (OH) -CH (OH) - (C = O) -O- -CH (OH) -CH (OH) - (C = O) -NH-, -S-maleimido - (CH2) 1- 6-, -S-maleimido- (C1-3 alkyl) - (C = O) -NH-, -S-maleimido- (C1-3 alkyl) - (C5-6 cycloalkyl) - ( C0-3 alkyl) -, - (C1-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -O-, - (C1-3 alkyl) - (C5- cycloalkyl) 6) - (C0-3 alkyl) - (C = O) -NH-, -S-maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) -, - (C0-3 alkyl) -phenyl - (C = O) -NH-, - (CH2) 1-12-NH- (C = O) -, - (CH2) 1-12- (C = O) -NH-, - (phenyl) - ( CH2) 1-3- (C = O) -NH-, -S- (CH2) - (C = O) -NH - (phenyl) -, - (CH2) 1-12- (C = O) -NH- (CH2) 1-12-, - (CH2) 2- (C = O) -O- (CH2) 2-O - (C = O) - (CH2) 2- (c = o) -NH-, - (C1-6 alkyl) - (C = O) -N- (C1.6 alkyl) -, acetal, ketal, acyloxyalkyl ether, -N = cH-, - (C1-6 alkyl) -SS- (C0-6 alkyl) -, - (C1-6 alkyl) -SS- (C1-6 alkyl) - (C = O) -O -, - (C1-6 alkyl) -SS- (C1-6 alkyl) - (C = O) -NH-, -SS- (CH2) 1-3- (C = O) -NH- (CH2) 1 -4- NH- (C = O) - (CH1) 1-3-, -SS- (C0-3 alkyl) - (phenyl) -, -SS- (C1-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) 1-5-, - (C1-3 alkyl) - (phenyl) - (C = O) - NH- (CH2) 1-5- (C = O) -NH-, -SS- (C1.3 alkyl) -, - (C1-3 alkyl) - (phenyl) - (C = O) -NH-, -O- (C1-C6 alkyl) -S (O2) - (C1 alkyl -6) -O- (C = O) -NH-, -SS- (CH2) 1-3- (C = O) -, - (CH2) 1-3- (C = O) -NH-N = CSS- (CH2) 1-3- (C = O) -NH- (CH2) 1-5-, - (CH2) 1-3- (C = O) -NH- (CH2) 1-5- (C = O) -NH-, - (CH2) 0-3- (heteroaryl) - (CH2) 0-3-, - (CH2) ü-3-phenyl- (CH2) ü-3-,

Y

image39

In another embodiment of compounds of formula (Ib), (IIb), (Illb), (IVb), or (Vb), the Sp2 spacer groups are selected from: -C1-C12 alkyl-, C3-C12 cycloalkyl, (- (CH2) 1-6-O- (CH2) 1-6-) 1-12-, (- (CH2) 1-4-NH- (CH2) 1-4) 1-12-, - (CH2) 1 -12O-, (- (CH2) 1-4-O- (CH2) 1-4) 1-12-O-, - (CH2) 1-12- (CO) -O-, - (CH2) 1- 12- (CO) -NH-, - (CH2) 1-12-O- (CO) -, - (CH2) 1-12-NH- (CO) -, (- (CH2) 1-4-O- (CH2) 1-4) 1-12-O- (CH2) 1-12-, - (CH2) 1-12- (CO) -O- (CH2) 1-12-, - (CH2) 1-12 - (CO) -NH- (CH2) 1-12-, - (CH2) 1-12-O- (CO) - (CH2) 1-12-, - (CH2) 1-12-NH- (CO) - (CH2) 1-12-, - (C3-C12 cycloalkyl) -, - (C1-C8 alkyl) - (C3-C12 alkyl) -, - (C3.C12 alkyl) - (C1.8 alkyl) - and - (C1-8 alkyl) - (C3-C12 cycloalkyl) - (C1.8 alkyl) -.

Although the Sp2 spacer group, when present, is considered attached to the biologically active agent, A, for the purpose of synthesizing the compounds of formula (Ib) or their sub-embodiments (IIb), (IIIb), (IVb) and ( Vb), the Sp2 spacer group, or a precursor thereof, can be introduced into the molecule to form the Z bond followed by the formation of the A-Sp2 bond. In this regard, some Sp2 spacer groups can be considered as bifunctional agents that have both a group capable of reacting with a reactive group X of compounds (la),

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(Ila), (Illa), (IVa), or (Va) to form a group Z, as a group capable of reacting with a group present in a biologically active agent, thus forming molecules of the form (A (Sp2) pZ - (Sp1) n) aL- (K) b where p is 1. A variety of bifunctional reagents that can be used as a source of spacer groups Sp1 and Sp2 are available from commercial suppliers including Pierce Biotechnology, Inc., (Rockford, IL , United States), (see, for example, Table II).

Table II

3 - ([2-Aminoethyl]) dithio) -propionic acid H- (a-Maleimidoacetoxy) -succinimide ester HCl

1.4- S / s-Maleimidobutane

1.4- S / s-Maleimidyl-2,3-dihydroxybutane S / s-Maleimidohexane S / s-Maleimidoethane

W-p-maleimidopropionic acid

W- (p-maleimidopropionic acid) hydrazide TFA

W- (p-Maleimidopropyloxy) succinimide ester

1,8-S / s-Maleimidotriethylinglycol

1.11 -S / s-Maleimidotetraethylene glycol

S / s (2- [succinimidoxycarbonyloxy] ethyl) sulfone

S / s (sulfosuccinimidil) suberate

C6-Succinimidyl 4-hydrazinonicotinate acetone hydrazone

C6-succinimidyl 4-formylbenzoate

1-5-Difluoro-2,4-dinitrobenzene

Dimethyl adipimidate-2HCl

Dimethyl pimelimidate-2HCl

Dimethyl Suberimidate 2HCl

1.4- Di- (3 '- [2'pyridyldithio] propionamido) butane Disuccinimidyl glutarate

Dithiobis (succimidylpropionate) (Lomant reagent)

Disuccinimidil suberate

Disuccinimidil tartarate

Dimethyl 3,3'-dithiob / spropionimidate-2HCl

Dithiob / S-Maleimidoethane

3,3'-Dithiob / s (sulfosuccinimidylpropionate)

Ethylene glycol b / s (succinimidylsuccinate)

W- £ -maleimidocaproic acid W- (£ -maleimidocaproic acid) hydrazide N- (£ -Maleimidocaproyloxy) succinimide ester W- (Y-Maleimidobutyryloxy) succinimide ester 1,6-Hexane-b / s-vinylsuflone WK-maleimidoundecanoic acid W- (K-maleimidoundecanoic acid) hydrazide

Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6-amidocaproate)

Succinimidyl 6- (3 '- [2-pyridyldithio] propionamido) hexanoate

m-Maleimidobenzoyl-W-hydroxysuccinimide ester

4- (4-N-Maleimidophenyl) -butyric acid hydrazideHCl

Methyl W-succinimidyl adipate

lysinyl] ethyl methanethiosulfate

3- 2 (2-Pyridyldithio) propionylhydrazide N- (p-Maleimidophenyl) isocyanate

Succinimidyl 4-hydrazinonicotinate acetone hydrazone

W-Succinimidil S-acetylthioacetate

W-Succinimidyl S-acetylthioproprionate

Succinimidyl 3- (bromoacetamido) propionate

Succinimidyl 4-formylbenzoate

W-succinimidyl iodoacetate

W-Succinimidyl (4-iodoacetyl) aminobenzoate

Succinimidyl 4- (W-maleimidomethyl) cyclohexane-1-carboxylate

Succinimidyl 4- (p-maleimidophenyl) butyrate

Succinimidyl 6- (p-maleimidopropionamido) hexanoate

4- Succinimidyloxycarbonyl-methyl-a- (2-pyridyldithium) toluene Succinimidyl- (4-psoralen-8-yloxy) butyrate W-Succinimidyl 3- (2-pyridyldithio) propionate Sulfodisuccinimidyl tartarate

Ethylene glycol b / s (sulfosuccinimidyl succinate)

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W- (£ -Maleimidocaproyloxy) sulphosuccinimide ester W- (Y-Maleimidobutryloxy) sulfosuccinimide ester W- (K-Maleimidoundecanoyloxy) sulfosuccinimide ester Sulfosuccinimidyl 6- (a-methyl-a- [2-pyridyldithio) hexolimido-6-thioamido) -toluamido-6-thioamidyl (6-pyrimidyl) -toluamido (6-pyrimidyl) -toluamido ester 3 '[2-pyridyldithio] propionamido) hexanoate m-Maleimidobenzoyl-W-hydroxysulfosuccinimide ester Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate Sulfosuccinimidyl 4- (W-maleimidomethyl) cyclohexane-1-carboxylate Sulfosuccinimidyl (pyrosuccinimidyl) butyl (pyrosuccinimidyl) butyl (pyrosuccinimidyl) pyrosuccinimidyl (4) eT rifluoracetylcaproyloxy) succinimide ester p- (tris [hydroxymethyl] phosphine) propionic acid (betaine)

Tr / s- (2-Maleimidoethyl) amine (Trifunctional)

Tr / s- (succimimidyl aminotricetate) (Trifunctional)

In those embodiments of compounds of the formulas (la) to (Vb) where n is 1, a spacer group - (Sp1) - is present; and in those embodiments where n is 0, the spacer group Sp1 is not present, and -Sp1- represents a covalent bond. Similarly, in some embodiments of compounds of the formulas (lb), (IIb), (lllb), (IVb), or (Vb), both n and p are 0, and the spacer groups Sp1 and Sp2 represent a bond covalent In other embodiments of compounds of the formulas (lb), (IIb), (lllb), (IVb), and (Vb), both n and p are 1, or alternatively n is 1 and p is 0, or as alternative n is 0 and p is 1.

In some compounds of formulas (la) through (Vb), the Sp1 or Sp2 spacer groups, when present, comprise a hydrolytically susceptible bond or an enzymatically degradable bond. In some embodiments of the present invention, the hydrolytically susceptible bonds or enzymatically degradable bonds present in Sp1 or Sp2 are independently selected from the group consisting of carboxylic acid esters, phosphate esters, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, disulfides, amides. In another embodiment of compounds of formulas (la) to (Vb), the Sp1 and Sp2 spacer groups, when present, comprise a hydrolytically susceptible bond or an enzymatically degradable bond comprising amino acids, peptides, polypeptides, nucleic acids and oligonucleotides. In another embodiment, the hydrolytically susceptible or enzymatically degradable bonds are selected from the group consisting of carboxylic acid esters, phosphate esters, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, disulfides, some amides, peptides, polypeptides, nucleic acids and oligonucleotides, each of which has a (C1-C6) alkyl group at both ends of the susceptible bond. In another embodiment, the hydrolytically susceptible or enzymatically degradable bonds present in the Sp1 and Sp2 spacers have (C1-C6) alkyl groups at each end, and Sp1 and Sp2 are selected from the group consisting of - (C1-C6 alkyl) - ( C = O) -O- (C1-C6 alkyl) -, - (C0-C6 alkyl) - PO4 (H) - (C0-C6 alkyl) -, or a salt thereof, - (C1-C6 alkyl) -SS- (C1-C6 alkyl) -, - (C1-C6 alkyl) - (C = O) -N- (C1-C6 alkyl) - or - (C1-C6 alkyl) -C = N- (C1 alkyl -C6) -. In another embodiment, the Sp1 and Sp2 bonds are selected from the group consisting of - (C1-C6 alkyl) - (aryl) -C = N- (C1-C6 alkyl) - and - (C1-C6 alkyl) -C = N- (aryl) - (C1-C6 alkyl) -. The person skilled in the art will recognize that many hydrolytically susceptible bonds are also enzymatically degradable bonds. For the purposes of this description, an enzymatically degradable bond can be placed in any position where a hydrolytically susceptible bond can be placed.

The Z group itself can also be a hydrolytically susceptible or enzymatically degradable bond. In one embodiment, the group Z may be selected from an amide, an ester or an imine, particularly an imine bearing an adjacent aryl group (i.e., a Schiff base). In another embodiment, Z may be a carboxylic acid ester, or an amide of a carboxylic acid.

In some embodiments of compounds (lb), (IIb), (lllb), (IVb) and (Vb), the active agent, A, can be released when bound by a hydrolytically susceptible bond. In an embodiment where p is 0, the link between A and Z is a hydrolytically susceptible or enzymatically degradable bond, which upon cleavage releases A (for example, Z is a hydrolytically susceptible or enzymatically degradable bond). In another embodiment, where p is 1, A is attached to its Sp2 spacer group by a hydrolytically susceptible or enzymatically degradable bond, whose cleavage releases A. In some embodiments where p is 1, the Sp2 spacer group comprises a bond or group hydrolytically susceptible or enzymatically degradable, whose cleavage releases A together with a covalently bonded group derived from Sp2. In one embodiment where p is 0 and A is a protein or polypeptide, A is directly linked to Z by a hydrolytically susceptible or enzymatically degradable bond between A and Z. In another embodiment, where p is 1 and A is a protein or polypeptide, A binds to its Sp2 spacer group via a hydrolytically susceptible or enzymatically degradable link between the Sp2 and A spacer group. In yet another embodiment where p is 0 and A is a protein or polypeptide having an amino acid of synthetic origin , A is directly linked to Z by a hydrolytically susceptible or enzymatically degradable bond between Z and the amino acid of synthetic origin in A. In yet another embodiment, where p is 1 and A is a protein or polypeptide having an amino acid of synthetic origin, A is linked to its Sp2 spacer group by a hydrolytically susceptible or enzymatically degradable link between the Sp2 spacer group and the amino acid of synthetic origin. acid in A.

The biologically active agents indicated by the group "A" of formula (IV) can be broadly selected. In some embodiments, biologically active agents can be selected from drugs, vaccines,

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antibodies, antibody fragments, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, tRNA tRNA, RNAi, DNA, cDNA, antisense constructions, ribozymes, etc., and combinations thereof). In one embodiment, the biologically active agents can be selected from proteins, peptides, polypeptides and combinations thereof. In another embodiment, the biologically active agents can be selected from nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, tRNA, tRNA, mRNA, DNA, cDNA, antisense constructs, ribozymes, etc., and combinations thereof). In another embodiment, the biologically active agents can be selected from steroids, lipids, fats and combinations thereof.

In a particularly useful embodiment, the biologically active agent is a therapeutic protein. Numerous therapeutic proteins are described throughout the application, such as, and not limited to, erythropoietin, granulocyte colony stimulating factor (G-CSF), GM-CSF, alpha interferon, beta interferon, human growth hormone, and imiglucerase .

In one embodiment, the biologically active agents may be selected from biologically active agents of specifically identified polysaccharides, proteins or peptides, including, but not limited to: agalsidase, alefacept, aspariginase, amdoxovir (DAPD), antide, becaplermin, botulinum toxin, including types A and B and lower molecular weight compounds with activity of botulinum toxin, calcitonins, cyanovirin, denileucine diftitox, erythropoietin (EPO), EPO agonists, alpha dornase, erythropoiesis stimulating protein (NESP), coagulation factors such as Factor V, Factor VII, Factor VlIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, von Willebrand factor; ceredasa, cerezima, alpha-glucosidase, collagen, cyclosporine, alpha defensins, beta defensins, desmopressin, exendin-4, cytokines, cytokine receptors, granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), alpha inhibitor -1 proteinase, elcatonin, granulocyte colony stimulating factor (GM-CSF), fibrinogen, filgrastim, growth hormones, human growth hormone (hGH), somatropin, growth hormone releasing hormone (GHRH), GRO-beta , GRO-beta antibody, bone morphogenic proteins such as bone morphogenic protein 2, bone morphogenic protein 6, OP-1; acid fibroblast growth factor, basic fibroblast growth factor, CD-40 ligand, heparin, human serum albumin, low molecular weight heparin (LMWH), alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, consensus interferon; interleukins and interleukin receptors such as interleukin-1 receptor, interleukin-2, interleukin-2 fusion proteins, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6 , interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17 receptor; lactoferrin and lactoferrin fragments, luteinizing hormone releasing hormone (LHRH), insulin, pro-insulin, insulin analogues, amylin, C-peptide, somatostatin, somatostatin analogs, including octreotide, vasopressin, follicle stimulating hormone (FSH), imiglucerase, vaccine against influenza, insulin growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF), plasminogen activators such as alteplase, urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, and teneteplase; Neural growth factor (NGF), osteoprotegerin, platelet-derived growth factor, tissue growth factors, transforming growth factor-1, vascular endothelial growth factor, leukemia inhibitor factor, keratinocyte growth factor (KGF), factor of glial growth (GGF), T lymphocyte receptors, CD molecules / antigens, tumor necrosis factor (TNF) (e.g., TNF- and TNF-p), TNF receptors (e.g., TNF-a receptor and TNF receptor -p), CTLA4, CTLA4 receptor, monocyte chemoattractant protein 1, endothelial growth factors, parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1, rasburicase, thymosin alpha 1 IIb / IIIa inhibitor, thymosin beta 10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase compounds (PDE), VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates, respiratory syncytial virus antibody, transmembrane regulatory gene cystic fibrosis ana (CFTR), deoxyribonuclease (Dnase), bactericidal / permeability enhancer protein (BPI), and anti-CMV antibody. Examples of monoclonal antibodies include etanercept (a dimeric fusion protein consisting of the extracellular ligand binding portion of the 75 kD human TNF receptor bound to the Fc portion of IgG1), abciximab, adalimumab, afelimomab, alemtuzumab, antibody against B lymphocyte, atlizumab, basiliximab, bevacizumab, biciromab, bertilimumab, CDP-484, CDP-571, CDP-791, CDP-860, CDP-870, cetuximab, clenoliximab, daclizumab, eculizumab, edrecolomabolumab, epumaumabomabolumabiz, bizumabizumabibumaboluma, font , gemtuzumab ozogamicin, ibritumomab tiuxetan, infliximab, inolimomab, keliximab, labetuzumab, lerdelimumab, olizumab, lym-1 radiolabelled metelimumab, mepolizumab, mitumomab, muromonad-CD3, nebacumab, natalizumab, odulimomab, omalizumab, oregovomab, palivizumab, pemtumomab, pexelizumab, rhuMAb-VEGF, rituximab, satumomab pendetide, sevirumab, siplizumab, tositumomab, I131 tositumomab, trastuzumab, tuvirumab, visilizumab, and fragments and mimetics thereof.

In one embodiment, the biologically active agent is a fusion protein. For example, and without limitation, the biologically active component may be an immunoglobulin or portion of an immunoglobulin fused to one or more specific useful peptide sequences. For example, the biologically active agent may contain an antibody Fc fragment. In one embodiment, the biologically active agent is a fusion protein of CTLA4. For example, the biologically active agent may be an Fc-CTLA4 fusion protein.

In a particularly useful embodiment, the biologically active agent is a human protein or a human polypeptide, for example, a human protein or a heterologously produced human polypeptide. Numerous

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Proteins and polypeptides are described herein, for which there is a corresponding human form (ie, the protein or peptide is normally produced in human cells in the human body). Therefore, in one embodiment, the biologically active agent is the human form of each of the proteins and polypeptides described herein for which there is a human form. Examples of such human proteins include, without limitation, human antibodies, human enzymes, human hormones and human cytokines such as granulocyte colony stimulating factor, macrophage and granulocyte colony stimulating factor, interferons (eg, alpha interferons and beta interferons ) and erythropoietin.

Other examples of therapeutic proteins that can serve as biologically active agents include, without limitation, factor VIII, factor removed from domain b VIII, factor VIIa, factor IX, anticoagulants; hirudin, alteplase, tpa, reteplasa, tpa, tpa - 3 out of 5 domains removed, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogues, hgh, glucagones, tsh, follitropin-beta, fsh, gm- csf, pdgh, ifn alfa2, ifn alfa2a, ifn alfa2b, inf-apha1, ifn-beta, inf-beta 1b, ifn-beta 1a, ifn-gamma (for example, 1 and 2), il-2, il-11 , hbsag, ospa, murine mab directed against the t-lymphocyte antigen, murine mab directed against tag-72, tumor associated glycoprotein, fab fragments derived from chimeric mab directed against the platelet surface receptor gpII (b) / III (a) , murine mab fragment directed against the tumor associated antigen ca125, murine mab fragment directed against the human carcinoembryonic antigen, cea, murine mab fragment directed against human cardiac myosin, murine mab fragment directed against the psma tumor surface antigen, murine mab fragments (fab / fab2 mix) directed against hmw-maa, fragment of mab mur ino (fab) directed against carcinoma associated antigen, mab (fab) fragments directed against nca 90, a non-specific surface granulocyte cross-reaction antigen, chimeric mab directed against the cd20 antigen found on the surface of lymphocytes b, humanized mab directed against the alpha chain of the il2 receptor, chimeric mab directed against the alpha chain of the il2 receptor, chimeric mab directed against tnf-alpha, humanized mab directed against an epitope on the surface of the respiratory syncytial virus, humanized mab directed against her 2, receptor 2 of the human epidermal growth factor, human mab directed against the antigen associated with anti-ctla 4 cytokeratin tumor, mab directed against the surface antigen of 20 dornase-alpha dnase lymphocyte, beta glucocerebrosidase, beta glucocerebrosidase, tnf-alpha, protein il-2-diphtheria toxin fusion, tnfr-lgg laronidase fragment fusion protein, dnaases, alefacept, darbepoietin alfa (factor e colony stimulant), tositumomab, murine mab, alemtuzumab, rasburicase, beta agalsidase, teriparatide, parathyroid hormone derivatives, adalimumab (lgg 1), anakinra, biological modifier, nesiritide, human natriuretic peptide b (hbnp), colony stimulating factors pegvisomant, human growth hormone receptor antagonist, recombinant activated protein c, omalizumab, immunoglobulin blocker e (lge), lbritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigment hormones, erythropoedin luteinizing, chorionic gonadotropin, hypothalamic releasing factors, ethanorcept, antidiuretic hormones, prolactin and thyroid stimulating hormone.

Examples of therapeutic antibodies that can serve as biologically active agents include, but are not limited to, HERCEPTIN ™ (Trastuzumab) (Genentech, CA) which is a humanized anti HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO ™ (abciximab) (Centocor) which is an anti-glycoprotein IIb / IIIa receptor in platelets for the prevention of clot formation; ZENAPAX ™ (daclizumab) (Roche Pharmaceuticals, Switzerland) which is a humanized immunosuppressive anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection; PANOREX ™ which is an anti-17-IA murine cell surface antigen IgG2a antibody (Glaxo Wellcome / Centocor); BEC2 which is a murine anti-idiotype IgG antibody (GD3 epitope) (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN ™ which is a humanized anti-aVp3 integrin antibody (Applied Molecular Evolution / Medlmmune); Campath; Campath 1H / LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab / Kanebo); RITUXAN ™ which is an anti-CD20 chimeric IgG1 antibody (IDEC Pharm / Genentech, Roche / Zettyaku); LYMPHOCIDE ™ which is a humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primate anti-CD80 antibody (IDEC Pharm / Mitsubishi); ZEVALIN ™ is a radiolabeled murine anti CD20 antibody (IDEC / Schering AG); IDEC-13l is a humanized anti-CD40L antibody (IDEC / Eisai); IDEC-151 is a primed anti-CD4 antibody (IDEC); IDEC-152 is a primed anti-CD23 antibody (IDEC / Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-factor 5 (CS) humanized antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-a antibody (CATIBASF); CDP870 is a humanized anti-TNF-a Fab fragment (Celltech); IDEC-151 is a primitive anti-CD4 IgG1 antibody (IDEC Pharm / SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex / Eisai / Genmab); CDP571 is a humanized anti-TNF-a IgG4 antibody (Celltech); LDP-02 is a humanized anti-a4p7 antibody (LeukoSite / Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA ™ is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN ™ is a humanized anti-VLA-4 IgG antibody (Elan); CAT-152, a human anti-TGF-p2 antibody (Cambridge Ab Tech); Cetuximab (BMS) is a monoclonal anti-EGF receptor antibody (EGFr); Bevacizuma (Genentech) is a human anti-VEGF monoclonal antibody; Infliximab (Centocore, JJ) is a chimeric monoclonal antibody (mouse and human) used to treat autoimmune disorders; Gemtuzumab ozogamycin (Wyeth) is a monoclonal antibody used for chemotherapy; and Ranibizumab (Genentech) is a chimeric monoclonal antibody (mouse and human) used to treat macular degeneration.

The conjugates of the invention and the compositions (for example, pharmaceutical compositions) containing

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Conjugates of the invention can be used to treat a variety of conditions. For example, there are many conditions for which treatment therapies are known to those skilled in the art in which biologically active agents are employed, as described herein. The invention contemplates that the conjugates of the invention (for example, polymer containing phosphorylcholine conjugated to a biologically active agent) and the compositions containing the conjugates of the invention can be used to treat said conditions and that said conjugates provide a treatment therapy enhanced relative to the same biologically active agent not coupled to a phosphorylcholine-containing polymer.

Therefore, the invention contemplates the treatment of a condition that is known to be treatable by a certain biologically active agent by treating the condition using the same certain biologically active agent conjugated to a phosphorylcholine-containing polymer. For example, erythropoietin conjugated to a polymer containing phosphorylcholine can be used to treat human conditions such as anemia and kidney disease (e.g., chronic renal failure) and, for example, G-CSF conjugated to a polymer containing phosphorylcholine can be used to Treat cancer patients receiving chemotherapy.

One skilled in the art may determine the amount of a conjugate of the invention that produces a therapeutic effect by repeated administration of increasing amounts of the conjugate (eg, pharmaceutical composition containing the conjugate) to achieve a clinically desired titration criteria.

Proteins and peptides for use as biologically active agents as described herein can be produced by any useful method that includes production by in vitro synthesis and by production in biological systems. Typical examples of in vitro synthesis methods that are well known in the art include solid phase synthesis ("SPPS") and condensation of solid phase fragments ("SPFC"). Biological systems used for protein production are also well known in the art. Bacteria (for example, E coli and Bacillus sp.) And yeast (for example, Saccharomyces cerevisiae and Pichia pastoris) are widely used for the production of heterologous proteins. In addition, heterologous gene expression for the production of biologically active agents for use as described herein can be carried out using animal cell lines such as mammalian cell lines (eg, CHO cells). In a particularly useful embodiment, the biologically active agents are produced in transgenic or cloned animals such as cows, sheep, goats and birds (eg, chicken, quail, ducks and turkeys), each as understood in the art. See, for example, U.S. Patent No. 6,781,030, issued August 24, 2004.

Biologically active agents such as proteins produced in domesticated birds, such as chickens, may be referred to as biologically active agents "derived from birds" (eg, therapeutic proteins derived from birds). The production of therapeutic proteins derived from birds is known in the art and is described in, for example, US Patent No. 6,730,822, issued on Tuesday, May 4, 2004.

In embodiments where the biologically active agent is a protein or polypeptide, the functional groups present in the amino acids of the protein polypeptide sequence can be used to bind the agent to the Z group, either directly via a covalent bond, or indirectly through a Sp2 spacer group. Bindings to biologically active protein or polypeptide agents can be made to naturally occurring amino acids in their sequence or to naturally occurring amino acids that have been added to the sequence or inserted instead of another amino acid. Biologically active protein or polypeptide agents may also comprise amino acids of synthetic origin in addition to common naturally occurring amino acids found in proteins and polypeptides. In addition to being present in order to alter the properties of a polypeptide or protein, amino acids of synthetic origin can be introduced to provide a functional group that can be used to bind the protein or polypeptide directly to the Z group, or indirectly through a Sp2 spacer group attached to the active agent. In addition, naturally occurring amino acids, for example, cysteine, can be used in this way.

Amino acids of synthetic origin can be introduced into proteins and peptides by a variety of means. Some of the techniques for the introduction of amino acids of synthetic origin are discussed in US Patent No. 5,162,218. First, amino acids of synthetic origin can be introduced by chemical modification of a polypeptide or protein in the amino acid side chain or in the amino or carboxyl end. Non-limiting examples of chemical modification of a protein or peptide could be methylation by agents such as diazomethane, or the introduction of acetylation into an amino group present in the lysine side chain or at the amino end of a peptide or protein. Another example of the modification of the amino group of protein / polypeptide to prepare an amino acid of synthetic origin is the use of methyl 3- mercaptopropionimidate ester or 2-iminothiolane to introduce a thiol carrier functionality (sulfhydryl, -SH) linked to positions in a protein or polypeptide that carries a primary amine. Once introduced, said groups can be used to form a covalent bond to the protein or polypeptide. The use of bifunctional agents, such as 2-iminothiolane, can be considered as the addition of a Sp2 group to a biologically active agent if the introduced group is used to form a covalent bond to Z.

Second, amino acids of synthetic origin can be introduced into proteins and polypeptides during

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chemical synthesis Synthetic methods are typically used to prepare polypeptides that have less than about 200 amino acids, that usually have less than about 150 amino acids, and more usually have 100 or less amino acids. Shorter proteins or polypeptides that are less than about 75 or less than about 50 amino acids can be prepared by chemical synthesis.

Synthetic preparation methods that are particularly convenient for allowing the insertion of unnatural amino acids in a desired location are known in the art. Suitable synthetic polypeptide preparation methods can be based on Merrifield solid phase synthesis methods where amino acids are added sequentially to a growing chain (Merrifield (1963) J. Am. Chem. Soc. 85: 2149-2156) . Automated systems for synthesizing polypeptides by such techniques are now commercially available from suppliers such as Applied Biosystems, Inc., Foster City, Calif. 94404; New Brunswick Scientific, Edison, N.J. 08818; and Pharmacia, Inc., Biotechnology Group, Piscataway, N.J. 08854.

Examples of synthetic amino acids that can be introduced during chemical synthesis of polypeptides include, but are not limited to: D-amino acids and mixtures of forms D and L of the 20 naturally occurring amino acids, N-formyl glycine, ornithine, norleucine , hydroxyproline, beta-alanine, hydroxivaline, norvaline, phenylglycine, cyclohexylalanine, t-butylglycine (t-leucine, 2-amino-3,3-dimethylbutanoic acid), hydroxy-t-butylglycine, amino butyric acid, cycloleucine, 4-hydroxyproline , pyroglutamic acid (5-oxoproline), azetidine carboxylic acid, pipecolinic acid, indolin-2-carboxylic acid, tetrahydro-3-isoquinolin carboxylic acid, 2,4-diaminobutyric acid, 2,6-diaminopimelic acid, 2,4- diaminobutyric acid, 2,6-diaminopimelic acid, 2,3-diaminopropionic acid, 5-hydroxylysine, neuraminic acid, and 3,5-diiodothyrosine.

Third, amino acids of unnatural origin can be introduced through biological synthesis in vivo or in vitro by inserting a nonsense codon (for example, an amber or ocher codon) into a DNA sequence (for example, the gene) that encodes the polypeptide in the codon corresponding to the position where the amino acid of synthetic origin is to be inserted. Such techniques are discussed, for example, in United States Patents No. 5,162,218 and 6,964,859, the descriptions of which are incorporated in their entirety herein by reference. A variety of methods can be used to insert the mutant codon, including oligonucleotide-directed mutagenesis. The altered sequence is transcribed and subsequently translated, in vivo or in vitro, into a system that provides a suppressor tRNA, directed against the nonsense codon that has been chemically or enzymatically acylated with the desired amino acid of synthetic origin. The synthetic amino acid will be inserted at the location corresponding to the nonsense codon. For the preparation of larger and / or glycosylated polypeptides, recombinant preparation techniques of this type are usually preferred. Among the amino acids that can be introduced in this way are: formyl glycine, fluoroalanine, 2-amino-3-mercapto-3-methylbutanoic acid, homocysteine, homoarginine and the like.

When amino acids of synthetic origin have a functionality that is susceptible to selective modification, they are particularly useful for forming a covalent bond to the protein or polypeptide. Circumstances in which a functionality is subject to selective modification include those in which the functionality is unique or where other functionalities that could react under the conditions of interest are prevented, either stereochemically or otherwise.

In another embodiment, the biologically active agents can also be selected from specifically identified drugs or therapeutic agents, including, but not limited to: tacrine, memantine, rivastigmine, galantamine, donepezil, levetiracetam, repaglinide, atorvastatin, alefacept, tadalafil, vardenafil, sildenafil, fosamprenavir, oseltamivir, valacyclovir and valganciclovir, abarelix, adefovir, alfuzosin, alosetron, amifostine, amiodarone, aminocaproic acid, aminohippurate sodium, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amlodipine, amsacrine, anagrelide, anastrozole, aprepitant, aripiprazole, asparaginase, atazanavir, atomoxetine, anthracyclines, bexarotene, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, cabergoline, capecitabine, carboplatin, carmustine, chlorambucine, sodium cilastatin, cisplatin, cladribine, chlodronate, cyclophosphamotide, cistrotidotamide retinoi acid co all trans; dacarbazine, dactinomycin, daptomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, doxorubicin, dutasteride, eletriptan, emtricitabine, enfuvirtide, eplerenone, epirubicin, estramustine, ethinyl estradiol, etoposide, exemestane, ezetimibe, fentanyl, fexofenadine, fludarabine, fludrocortisone , fluorouracil, fluoximasterone, flutamide, fluticazone, fondaparinux, fulvestrant, gamma-hydroxybutyrate, gefitinib, gemcitabine, epinephrine, L-Dopa, hydroxyurea, icodextrin, idarubicin, ifosfamide, imatinib, itineconazole, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinotezolidene , levamisole, lisinopril, lovotiroxina sodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, memantine, mercaptopurine, mequinol, bitartrate metaraminol, methotrexate, metoclopramide, mexiletine, miglustat, mitomycin, mitotane, mitoxantrone, modafinil, naloxone, naproxen, nevirapine, nicotine , nilutamide, nitazoxanide, nitisinone, norethindrone, octreotide, oxaliplatin, palonosetron, pamidronate, pemetrexed, pergolide, pentostatin, pilcamicina, porfimer, prednisone, procarbazine, prochlorperazine, ondansetron, palonosetron, oxaliplatin, raltitrexed, rosuvastatin, sirolimus, streptozocin, pimecrolimus, sertaconazole, tacrolimus, tamoxifen, tegaserod, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, tiotropium, topiramate, topotecan, treprostinil, tretinoin, valdecoxib, celecoxib, rofecoxib, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, voriconazole, dolasetron, granisetron, formoterol, fluticasone, leuprolide, midazolam, alprazolam, amphotericin B, podophyllotoxins, antivirals

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nucleosidase, aroyl hydrazones, sumatriptan, eletriptan; macrolides such as erythromycin, oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin, flurithromycin, dithromromycin, josamycin, spiromycin, midecamycin, loratadine, desloratadine, leucomycin, mithyamycin, swithiamycin; fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin, amicacin, kanamycin, neomycin, and streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin, colistimethate; polymyxins such as polymyxin B, capreomycin, bacitracin, penems; penicillins, including penicillinase sensitive agents such as penicillin G, penicillin V; penicillinase resistant agents such as methicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, naphcillin; active agents of gram negative microorganisms such as ampicillin, amoxicillin, and hetacillin, cilia, and galampicillin; antipseudomonas penicillins such as carbenicillin, ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporins such as cefpodoxime, cefprozil, ceftbutene, ceftizoxime, ceftriaxone, cephalothin, cefapirin, cephalexin, cefradrine, cefoxitin, cephamandol, cefazolin, cephaloridin, cefaclorus, cephadroxyl, cephaloximatrin, cephaloximethyl, cephalophatrin cephaphthiophyte, cephalophatrin cephaphthiophyte cefoperazone, cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams such as aztreonam; and carbapenems such as imipenem, meropenem, and ertapenem, pentamidine isethionate, albuterol sulfate, lidocaine, metaproterenol sulfate, beclomethasone diprepionate, triamcinolone acetamide, budesonide acetonide, salmeterol, ipratrium trichlorate, chroma trichlorate of ergotamine; taxanes such as paclitaxel; SN-38 and thyrostost. Biologically active agents can also be selected from the group consisting aminohippurate sodium, amphotericin B, doxorubicin, aminocaproic acid, aminolevulinic acid, aminosalicylic acid, bitartrate metaraminol, pamidronate disodium, daunorubicin, lovotiroxina sodium, lisinopril, cilastatin sodium, mexiletine, cephalexin, deferoxamine, and amifostine in another embodiment.

C. Pharmaceutical compositions

The present invention includes and provides pharmaceutical compositions comprising one or more compounds of the invention and one or more pharmaceutically acceptable excipients. The compounds of the invention may be present as a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, in the pharmaceutical compositions of the invention. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption retarding and isotonic agents, and the like, compatible With the pharmaceutical administration.

Pharmaceutically acceptable carriers for use in the formulation of compounds of formula (la) to (Vb) include, but are not limited to: solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, stearate. magnesium, stearic acid, and the like; and liquid vehicles such as syrups, saline, phosphate buffered saline, water and the like. Vehicles may include any delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax, ethyl cellulose, hydroxypropyl methylcellulose, methyl methacrylate, or the like.

Other fillers, excipients, saporifers and other additives such as those known in the art, may also be included in a pharmaceutical composition according to this invention. The use of such media and agents for pharmaceutically active substances is known in the art. Except in the event that any conventional agent or medium is incompatible with the active compound, the use thereof in the compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions of the present invention.

Pharmaceutical preparations include all types of formulations. In some embodiments, they are parenteral formulations (including subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrathecal, intraventricular, intracranial, intraspinal, intracapsular and intraosseous) suitable for injection or infusion (e.g., powders or concentrated solutions that can be reconstituted or diluted, as well as suspensions and solutions). When the composition is a solid that requires reconstitution or a concentrate that requires dilution with liquid media, any suitable liquid medium may be employed. Preferred examples of liquid media include, but are not limited to, water, saline, phosphate buffered saline, Ringer's solution, Hank's solution, dextrose solution, and 5% human serum albumin.

When a compound or pharmaceutical composition comprising a compound of formula (la) to (Vb) is suitable for the treatment of cell proliferative disorders, including, but not limited to, cancers, the pharmaceutical compound or composition can be administered to a subject through a variety of routes that include injection directly into tumors, bloodstream or body cavities.

Although the pharmaceutical compositions can be liquid solutions, suspensions or powders that can be reconstituted immediately before administration, they can also take other forms. In some embodiments, the pharmaceutical compositions may be prepared in the form of syrups, broths, bowls, granules,

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pastes, suspensions, creams, ointments, tablets, capsules (hard or soft), sprays, emulsions, microemulsions, patches, suppositories, powders, and the like. The compositions can also be prepared for routes of administration other than parenteral administration including, but not limited to, topical (including buccal and sublingual), pulmonary, rectal, transdermal, transmucosal, oral, ocular, etc.

In some embodiments, the pharmaceutical compositions of the present invention comprise one or more compounds of formula (Ib). In another embodiment, the pharmaceutical compositions of the present invention comprise one or more compounds of formula (11 b). In other embodiments, the pharmaceutical compositions of the present invention comprise one or more compounds of formula (IIIb), one or more compounds of formula (IVb), or one or more compounds of formula (Vb). In yet other embodiments, the pharmaceutical compositions of the present invention comprise compounds of the present invention that have a star polymer conjugated to a biologically active agent. In other embodiments, the pharmaceutical compositions comprise two or more compounds independently selected from formula (Ib), (IIb), (IIIb) or (IVb), or (Vb), In an alternative embodiment, the pharmaceutical compositions comprise compounds of formula ( Ib), (IIb), (IIIb) or (IVb), or (Vb) and one or more additional biologically active agents. In some embodiments, the pharmaceutical compositions comprise one or more compounds of formula (Ib), (IIb), (IIIb) or (IVb), or (Vb) in which the biologically active agent is selected from those biologically active polysaccharide agents , proteins or peptides identified specifically set forth in section B.3; or alternatively, in other embodiments, those specifically identified drugs or therapeutic agents set forth in section B.3.

The pharmaceutical compositions of the present invention may comprise a compound of the present invention having a biologically active agent selected from erythropoietin, granulocyte colony stimulating factor (G-CSF), alpha interferon, beta interferon, human growth hormone, and imiglucerase . In other embodiments, the pharmaceutical compositions comprise a compound of formula (IIb), (IIIb) or (IVb), or (Vb), where the biologically active agent is selected from erythropoietin, granulocyte colony stimulating factor (G-CSF) , interferon alfa, interferon beta, human growth hormone, and imiglucerase. In other embodiments of the invention, the pharmaceutical compositions comprise a compound of formula ((IIIb) or (Vb), wherein the biologically active agent is selected from erythropoietin, granulocyte colony stimulating factor (G-CSF), alpha interferon, interferon beta, human growth hormone, and imiglucerase.

Other pharmaceutical compositions of the present invention may comprise one or more compounds of the formulas (Ib), (IIb), (IIIb) or (IVb), or (Vb) that function as biological ligands that are specific to an antigen or target molecule . Such compositions may comprise a compound of formula ((IIIb) or (Vb), wherein the biologically active agent is a polypeptide comprising the amino acid sequence of an antibody, or an antibody fragment such as a FAb2 or FAb 'fragment or a variable antibody region Alternatively, the compound may be a compound of formulas (IIb) or (IVb), and the polypeptide may comprise the antigen binding sequence of a single chain antibody, when a biologically active agent present in a compound of formula (Ia) to (Vb) it functions as a specific ligand for an antigen or target molecule, these compounds can also be used as diagnostic reagents or in diagnostic assays.

The amount of a compound in a pharmaceutical composition will vary depending on several factors. In one embodiment, it may be a therapeutically effective dose that is suitable for a single dose container (eg, a vial). In one embodiment, the amount of the compound is an amount suitable for a single-use syringe. In yet another embodiment, the amount is suitable for multi-purpose dispensers (eg, containers suitable for the administration of drops of formulations when used to administer topical formulations). One skilled in the art may determine the amount of a compound that produces a therapeutically effective dose experimentally by repeated administration of increasing amounts of a pharmaceutical composition to achieve a clinically desired titration criteria.

Generally, a pharmaceutically acceptable excipient will be present in the composition in an amount of from about 0.01% to about 99.999% by weight, or from about 1% to about 99% by weight. The pharmaceutical compositions may contain from about 5% to about 10%, or from about 10% to about 20%, or from about 20% to about 30%, or from about 30% to about 40% %, or from about 40% to about 50%, or from about 50% to about 60%, or from about 60% to about 70%, or from about 70% to about 80%, or from about 80% to about 90% by weight of excipient. Other suitable ranges of excipients include from about 5% to about 98%, from about 15 to about 95%, or from about 20% to about 80% by weight.

Pharmaceutically acceptable excipients are described in a variety of known sources, including, but not limited to, "Remington: The Science & Practice of Pharmacy," 19th ed., Williams & Williams, (1995) and Kibbe, AH, Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, DC, 2000.

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D. Treatment methods

Methods for treating a condition sensitive to a biological agent comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable composition of the invention as described above are described herein. Dosage and administration are adjusted to provide sufficient levels of the biologically active agent or agents to maintain the desired effect. The dosage and / or appropriate administration protocol for any given subject may vary depending on several factors, including the severity of the pathology, the general health of the subject, age, weight and sex of the subject, diet, time and The frequency of administration. drug combinations), reaction sensitivities, and tolerance / response to therapy. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and criteria of the physician.

The pharmaceutical compositions described herein can be administered individually. Alternatively, two or more pharmaceutical compositions may be administered sequentially, or in a cocktail or combination containing two compounds of the formulas (Ib), (IIb), (IIIb), (IVb), or (Vb) or a compound of the formulas (Ib), (IIb), (IIIb) or (IVb), or (Vb) and other biologically active agent.

The treatment methods may employ a pharmaceutical composition comprising one or more compounds of formula (Ia) to (Vb). In other embodiments, the treatment will employ a pharmaceutical composition comprising one or more compounds of formula (Ib), (IIb), (IIIb), (IVb) or (Vb). In yet other embodiments, the treatment will employ a pharmaceutical composition comprising those compounds of the present invention that have a star polymer conjugated to a biologically active agent. In other embodiments, the treatment methods comprise administering two or more compounds selected independently of the formula (Ib), (IIb), (IIIb), (IVb), or (Vb). In yet another embodiment, the treatment method comprises administering one or more compounds of formula (Ib), (IIb), (IIIb) or (IVb) or (Vb) where the biologically active agent is selected from those biologically active polysaccharide agents , specifically identified proteins or peptides or those identified pharmacological or therapeutic agents specifically set forth in section B.3, above.

In some embodiments, the treatment methods comprise administering to a subject or patient in need thereof a pharmaceutical composition comprising a compound of the present invention that has a biologically active agent selected from the group consisting of: erythropoietin, granulocyte colony stimulating factor (G-CSF), interferon alfa, interferon beta, human growth hormone, and imiglucerase. In other embodiments of the invention, the pharmaceutical compositions comprise a compound of formula (Ib), (IIb), (IIIb), (IVb) or (Vb), wherein the biologically active agent is selected from the group consisting of: erythropoietin, granulocyte colony stimulating factor (G-CSF), interferon alfa, interferon beta, human growth hormone, and imiglucerase.

The use of erythropoietin for the treatment of anemia and oncology, granulocyte colony stimulating factor (G-CSF) in oncology and immunology applications, alpha interferon in oncology applications and in the treatment of hepatitis C, interferon beta In the treatment of multiple sclerosis, human growth hormone in the treatment of growth disorders, and imiglucerase for the treatment of Gaucher syndrome are well described in the art. Other uses of the biologically active agents set forth herein can be found in standard reference texts such as the Merck Manual of Diagnosis and Therapy, Merck & Co., Inc., Whitehouse Station, NJ and Goodman and Gilman's The Pharmacological Basis of Therapeutics, Pergamon Press, Inc., Elmsford, NY, (1990).

Administration of the compounds and pharmaceutical compositions of the present invention can be achieved by any suitable route, including, but not limited to, various forms of injection. Injection routes include subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrathecal, intraventricular, intracranial, intraspinal, intracapsular and intraosseous. Injectable formulations include, but are not limited to, ready-to-inject solutions, emulsions, suspension and reconstitutable dry powders that are diluted with an acceptable solvent before administration, and liquid concentrates for dilution before administration, among others. The compositions can also be administered by other routes including, but not limited to, topical (including buccal and sublingual), pulmonary, rectal, transdermal, transmucosal, oral, nasal, ocular, intrathecal, subcutaneous and intraarterial.

E. Methods of preparing compounds of the invention

Methods for preparing the compounds of the invention, such as compounds of the formulas (Ia) to (Vb), are described herein. The compounds may be prepared by any means known in the art for the preparation of polymeric compounds and their conjugates. Standard synthetic methods and procedures for the preparation of organic molecules and transformations and manipulations of functional groups, including the use of protecting groups, can be obtained from the relevant scientific literature or from standard reference textbooks in the field. Although not limited to one or several sources, recognized reference textbooks of organic synthesis include: Smith, M. B .; March, J., March's Advanced Organic Chemistry:

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Reactions, Mechanisms, and Structure, 5th ed .; John Wiley & Sons: New York, 2001; Greene, T.W .; Wuts, P.G. M., Protective Groups in Organic Synthesis, 3rd ed; John Wiley & Sons: New York, 1999; and Odian, G., Principles of Polymerization, 4th ed., Wiley-Interscience John Wiley & Sons: New York, 2004. The following descriptions of synthetic methods are designed to illustrate, but not limit, general procedures for the preparation of compounds of the invention.

E.1 Preparation of monomers.

The preparation of the polymeric portion of the compounds of the invention can be carried out by polymerization of suitable monomers in the form:

image40

where Q is H, methyl, or ethyl, T is H, -CH3, -CH2-CH3, and -CH2-CH2-O-PC, and PC represents a phosphorylcholine group. When T is hydrogen and Q is H or methyl, the monomers are acrylic acid and 2-methylacrylic acid, respectively, both of which are commercially available from suppliers that include Sigma-Aldrich. Ethyl methacrylate and methyl methacrylate are also available from Sigma-Aldrich. When T is hydrogen and Q is ethyl, the monomer is 2-ethylacrylic acid (EAA) that is commercially available or can be prepared from diethyl malonate or by the methods employed by Yong et al. in J. Polymer Sci 42: 3828-3835 (2004). Monomers in which T is -CH2-CH2-O-PC, including the internal salt of 2-methacryloxyxyethyl-2'-trimethylammonium-ethyl phosphate (also called hydroxyethylmetacryloyl phosphorylcholine, HEMA-PC or MPC), can be prepared as described in US 5,741,923 from 2-hydroxyethyl acrylic acid, 2-hydroxyethylmethyl acrylic acid or 2-hydroxyethyl acrylic acid.

E.2 Monomer composition

The polymer group present in the compounds of the formulas (la) to (Vb) is 2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl poly [phosphate] (or an internal salt thereof) prepared from phosphate of 2-Methacryloxyethyl-2'-trimethylammonium ethyl or a salt thereof (also called hydroxyethylmethacryloyl phosphorylcholine, HEMA-PC or MPC). In those embodiments in which one or more comonomers are polymerized with 2-methacryloxyethyl-2'-trimethylammonium-ethyl phosphate, the molar ratio of that zwitterionic monomer to the total amount of comonomers is in the range of 1:50 to 50: 1; In other embodiments, the ratio is in the range of about 1:10 to about 10: 1, and in yet other embodiments, the ratio is in the range of about 1: 5 to 1: 1. In another group of embodiments, the 2-methacryloxyethyl-2'-trimethylammonium-ethyl phosphate monomers comprise 100% or about 100%, or about 98%, about 95%, about 90%, about 85 %, about 80%, about 75%, about 70%, about 60% or about 50% of the total monomers present in any compound of formulas (la) to (Vb). When 100% of the polymer comprises ethyl 2-methacryloxyethyl-2'-trimethylammonium phosphate monomers (ie, HEMA-PC), the polymer portion ("polymer group") of the compounds (shown without the attached L or E groups )

image41

it is poly [2- (methacryloxyethyl) -2 '- (trimethylammonium) ethyl phosphate] (ie, poly-HEMA-PC). E.3 Radical polymerization and coupling to biologically active agents E.3.1 Live radical polymerizations / pseudo-radical radical polymerizations

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The compounds of the present invention can be prepared by various methods. A method for preparing the polymers of the invention includes live radical polymerization, which can be advantageously used to prepare compounds of formulas (Ia) through (Vb) that have specific architectures that include a controlled chain length and low polydispersity. Live radical polymerization is analyzed by Odian, G. in Principles of Polymerization, 4th ed., Wiley-lnterscience John Wiley & Sons: New York, 2004, and applied to zwitterionic polymers, for example, in US 6,852 .816. Several different living radical polymerization methodologies can be employed, including stable free radical polymerization (SFRP) and radical addition-fragmentation transfer (RAFT). In addition, atom transfer radical polymerization (ATRP), considered by some as a form of pseudo-living polymerization, provides a convenient method for the preparation of the compounds of the invention.

E.3.1.1 Initiators and compound architecture

The preparation of the compounds of the formula (la) e (Ib) by ATRP is plotted in Schemes IA and IB. The reaction involves radical polymerization of selected monomers that begin with an initiator that carries one or more halogens (eg, (X- (Sp1) n) aL-halogen (s)) provided by the group -L - ((Sp1 ) n- X) a (Scheme IA). The initiator is activated by a catalyst such as a transition metal salt that can be solubilized by a ligand (for example, bipyridine). Alternatively, an initiator carrying one or more halogens can provide a group L with suitable functionalities (for example, hydroxyl, amine, or carboxyl) for the subsequent addition of residues - (Sp1) nX (Scheme IB), which is particularly useful when a group - (Sp1) nX is introduced by a bifunctional agent.

IA and IB schemes

image42

IB scheme

* L-Br

+

Monomers

Catalyst (for example, CuBr)

(ligand for catalyst when used)

* L-Polymer-Br

(x-tspiu-

(X- (Sp 1) „) a-L - polymer-Br

compound of formula (la)

'indicates a binding functionality to join the residues X- (Sp1) - with L

Agent "A"

biologically

active

(A-Z- (Spl) „) a-L-polymer-Br

compound of formula (Ib)

Compounds that have a complex architecture can also be prepared by ATRP as depicted in the diagram in the two embodiments shown in Schemes IIA and IIB for branched and branched compounds.

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Schemes IIA and IIB IIA

image43

The compounds of formulas (Ia) and (Ib) include molecules that have complex architectures that include branched compounds that have multiple polymer arms including, but not limited to, comb and star structures. Comb architectures can be achieved using linear initiators that carry three or more halogen atoms, preferably the halogens are chlorine, bromine or iodine atoms, more preferably the halogens are chlorine or bromine atoms. Star architectures can also be prepared using compounds that carry multiple halogens in a single carbon atom (for example, trichloromethane or 2,2,2-trichloroethanol, etc.) or cyclic molecules that carry multiple halogens (for example, tri- or tetrabromocycloalkanes such as tribromocyclohexanol or tetrabromocyclohexanol). In some embodiments, compounds that have star architecture have 3 polymer arms and in other embodiments they have 4 polymer arms.

While the halogens set forth in Schemes I and II are indicated as bromine, other halogens, particularly chlorine, can be used. In some embodiments of compounds of formulas (Ia) to (Vb), including those having residues with polymer architectures in comb or star, bromine, chlorine and iodine halogens are preferred. In other embodiments, the halogens of compounds of the formulas (Ia) to (Vb), including those having moieties with comb or star polymer architectures, are bromine and chlorine.

In those embodiments in which an initiator is used that carries one or more halogens (for example, (X- (Sp1) n) aL-halogen (s)) in the preparation of compounds of the formulas (Ia) to (Vb) , L may have a group X or a protected form thereof. Alternatively, an initiator that carries one or more halogens can provide a group L with suitable functionalities (e.g., hydroxyl, amine, or carboxyl or protected forms thereof) that can be used to introduce a group - (Sp1) nX through of the use of a bifunctional agent (see, for example, Schemes IB and IIB).

A wide variety of initiators can be used to prepare compounds of the invention, including several

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initiators set forth in US 6,852,816. In some embodiments, the initiators used for aTrP reactions to prepare compounds of the invention are selected from alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, cyclic alkyl ethers and ethers bearing a halogen where unbranched compounds are prepared, and more than one halogen where branched molecules are prepared. In other embodiments, the initiators are selected from alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, ethers and cyclic alkyl ethers which carry a chlorine or bromine atom where unbranched compounds are prepared, or alternatively, more than one chlorine and / or bromine where branched molecules will be prepared.

The initiators used for the ATRP reactions can be hydroxylated. In some embodiments, the initiators used for the ATRP reactions to prepare compounds of the invention are selected from alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, cyclic alkyl ethers and ethers bearing a group hydroxyl and also carry a halogen where unbranched compounds will be prepared, or alternatively, more than one halogen where branched molecules will be prepared. In other embodiments, the initiators are selected from alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, cyclic alkyl ethers and ethers that carry a hydroxyl group and also carry a chlorine or bromine atom where unbranched compounds will be prepared, or alternatively, more than one chlorine and / or bromine where branched molecules will be prepared. In another embodiment in which bifurcated compounds of the invention will be prepared, the initiators are dihydroxy or trihydroxy, or even polyhydroxy alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, cyclic alkyl ethers and ethers which also carry a chlorine or bromine atom where unbranched compounds are prepared, or alternatively, more than one chlorine and / or bromine where branched and branched compounds will be prepared. When the initiators are hydroxylated, after the polymerization reaction to add the polymeric portion of the molecule, the hydroxyl group or groups can function as the "X" reactive group of the compounds of the formulas (Ia), (IIa), ( IIIa), (IVa) or (Va). Alternatively, after polymerization, the hydroxyl group or groups may be reacted with hydroxyl reactive bifunctional agents such as those set forth in Table II (for example, N- (p-maleimidophenyl) isocyanate) to prepare a compound of formula ( Ia), (IIa), (IIIa), (IVa) or (Va) with other reactive groups.

The initiators used for the ATRP reactions may have one or more amine groups. In some embodiments, the initiators used for ATRP reactions to prepare compounds of the invention are alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, cyclic alkyl ethers and ethers bearing an amine group and they also carry a halogen where unbranched compounds will be prepared, or alternatively, more than one halogen where branched molecules will be prepared. In other embodiments, the initiators are alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, cyclic alkyl ethers and ethers that carry an amine and also carry a chlorine or bromine atom where they will be prepared unbranched compounds, or alternatively, more than one chlorine and / or bromine where branched molecules will be prepared. In another embodiment in which bifurcated compounds of the invention will be prepared, the initiators are diamino or triamino, or even polyamine alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, cyclic alkyl ethers and ethers which also carry a chlorine or bromine atom where unbranched compounds are prepared, or alternatively, more than one chlorine and / or bromine where branched and branched compounds will be prepared. When the initiators carry one or more amine groups, after the polymerization reaction to add the polymer portion of the molecule, the amine group or groups can function as the "X" reactive group of the compounds of the formulas (Ia), ( IIa), (IIIa), (IVa) or (Va). Alternatively, after polymerization, the amine group or groups can be reacted with bifunctional agents reactive with amines such as those set forth in Table II (for example, 3,3'-dithiob / s (sulfosuccinimidyl propionate), sulfosuccinimidyl 4- (p- maleimidophenyl) butyrate, W- (£ -trifluoroacetylcaproxyl) succinimide ester, p- (tris [hydroxymethyl] phosphine) propionic acid, etc.) to prepare additional compounds of the formulas (Ia), (IIa), (IIIa), (IVa) or (Va).

Chlorinated or brominated alkylcarboxylic acids, which include alkyl dicarboxylic acids, substituted with amino or hydroxy groups can also be used as initiators. In some embodiments of the invention where ATRP is used to prepare the compounds of formulas (Ia) through (Vb), the initiators may be alkylcarboxylic acids bearing one or more halogens selected from chlorine and bromine. In some embodiments, when branched compounds are to be prepared, the initiators may be alkylcarboxylic acids bearing two or more carboxyl groups and a halogen selected from chlorine and bromine and two or more groups selected from -OH and -NH2. In embodiments in which branched compounds are to be prepared, the initiators may be alkylcarboxylic acids bearing two or more halogens selected from chlorine and bromine. In embodiments in which branched and branched compounds are to be prepared, the initiators may be alkylcarboxylic acids bearing two or more halogens selected from chlorine and bromine and two or more carboxyl groups. When the initiators carry one or more carboxyl groups, after the polymerization reaction to add the polymeric portion of the molecule, the carboxyl group or groups can function as the reactive group "X" of the compounds of the formulas (Ia), ( IIa), (IIIa), (IVa) or (Va). Alternatively, after polymerization, the carboxyl group or groups may be reacted with bifunctional agents reactive with carboxyl such as those set forth in Table II (for example, N- (s-Trifluoroacetylcaproyloxy) succinimide ester, 3- (HCl acid) [2-aminoethyl]) dithio) -propionic, etc.)

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to prepare additional compounds of the formulas (la), (IIa), (Illa), (IVa) or (Va).

Chlorinated or brominated alkanes substituted with two or more groups selected from -COOH, -OH and -NH2 can also be used as initiators for the preparation of bifurcated compounds in which ATRP is used to prepare compounds of formulas (Ia) to ( Vb). The initiators can be alkanes that carry a halogen selected from chlorine and bromine and two or more groups selected from -COOH, -OH and -NH2. In some embodiments in which branched and branched compounds are to be prepared, the initiators may be alkanes that carry two or more halogens selected from chlorine and bromine and two or more groups selected from -COOH, -OH and -NH2. When the initiators carry two or more groups selected from -COOH, -OH and -NH2, after the polymerization reaction to add the polymer portion of the molecule, the groups -COOh, -OH and -NH2 present can function as the groups X reagents of compounds of the formulas (Ia), (IIa), (IIIa), (IVa) or (Va). Alternatively, after polymerization, the -COOH, -OH and -NH2 groups can be reacted with reactive bifunctional agents of -COOH, -OH or -NH2 such as those set forth in Table II to prepare additional compounds of the formulas (Ia ), (IIa), (IIIa), (IVa) or (Va). In this way, asymmetric bifurcated compounds of the formulas (Ia), (IIa), (IIIa), (IVa) or (Va) can be prepared, especially when two different groups selected from -COOH, -OH and -NH2 are present in the initiator; subsequent reaction with one or more biologically active agents will produce asymmetric bifurcated compounds of the formulas (IBM), (Ibis), (Iamb), (Ivy) or (Vb).

In addition to the groups present in the initiators that are used in the formation of polymer chains (for example, Br or Cl) and those that serve to join X or X-Sp1 moieties, the imitators can also contain one or more groups - OH, amino, monoalkylamino, dialkylamino, -O-alkyl, -COOH, -COO-alkyl or phosphate independently selected (or protected forms thereof) that will be present as substituents in group L of compounds of formulas (Ia) to (Vb ).

A wide variety of initiators are available in the market, for example: 3-bromopropionaldehyde dimethyl acetal; 3-bromopropanamine; 3-bromopropionic acid; Bromobutanoic acid 4; 2,3-dibromopropionic acid and its methyl ester;

2,3-dichloropropionic acid and its methoxy ester; 2,3-dichloropropionamide; 2,2,3-trichloropropionamide; 2,3-dibromo-1- propanol; 1,3-dibromo-2-propanol; 1,3-dibromo-2-propanamine; 3-Bromo-2- (bromomethyl) propanoic acid, 2,2,3-trichloro-1-hydroxybutylformamide; 2,2,3,3,3-pentachloropropionamide; (2,2,3-trichloro-1-hydroxybutyl) -carbamic acid methyl ester; 2,3-dibromosuccinic acid; 3,4-dibromodihydro-2,5-furandione; Methyl 2,3-dibromo-2-methylpropanoate; 2,3-dibromo-4-oxobutanoic acid; Ethyl 2,3-dibromo-2-methylpropanoate; 2,3-dibromo-N, N-bis (hydroxymethyl) propanamide; 3,4-dibromo-4-phenyl-2-butanone; 2,3-dibromo-3-phenylpropanoic acid; 2,3,4-tribromobutanoic acid; 4,5-dibromo-1,2-cyclohexanedicarboxylic acid; 2,2-bis (bromomethyl) -1,3-propanediol; 3,5-dibromo-4-oxopentanoic acid; and bromoacetic acid N-hydroxysuccinimide ester available from Sigma-Aldrich (St. Louis, MO). Properly protected forms of these initiators can be prepared using standard methods in the art as necessary.

E.3.1.2 Catalyst and win!

The catalyst for use in ATRP or group radical transfer polymerizations may include salts.

suitable for Cu1 +, Cu2 +, Fe2 ++ Fe3 +, Ru2 + Ru.3 +, Cr2 +, Cr3 +, Mo2 +, Mo.3 +, W2 +, W3 +, Mn2 +, Mn2 +, Mn4 +, Rh3 +, Rh4.

Re2 +, Re3 +, Co1 +, Co.2 +, Co3 +, V2 +, V3 +, Zn.1 +, Zn2 +, Ni2 +, Ni3 +, Au1 +, Au2 +, Ag1 + and Ag2 +. Suitable salts include, but are not limited to: halogen, C1-C6 alkoxy, sulfates, phosphate, triflate, hexafluorophosphate, methanesulfonate, arylsulfonate salts. In some embodiments, the catalyst is a chloride salt, bromide of the metal ions mentioned above. In other embodiments, the catalyst is CuBr, CuCl or RuCl2.

2+

3+

2+

3+

2+

3+

2+

3+

2+

4+

3+

4+

In some embodiments, the use of one or more ligands to solubilize the transition metal catalyst is desirable. Suitable ligands are usefully used in combination with a variety of transition metal catalysts, including chloride or copper bromide, or transition metal salts of ruthenium chloride, which are part of the catalyst. The choice of a ligand affects the function of the catalyst since the ligands not only help solubilize transition metal catalysts in organic reaction media but also adjust their redox potential. The selection of a ligand is also based on the solubility and separability of the catalyst from the product mixture. When the polymerization is to be carried out in a liquid phase, soluble ligands / catalysts are generally desirable, although immobilized catalysts can be employed. Suitable ligands include those pyridyl groups (including alkyl pyridines, for example, 4.4, dialkyl-2,2'-bipyridines) and pyridyl groups bearing an imino group substituted with alkyl, when present, longer alkyl groups provide solubility in mixtures of less polar monomers and solvent media. Triphenylphosphines and other phosphorus ligands, in addition to indanyl or cyclopentadienyl ligands, can also be used with transition metal catalysts (for example, Ru + 2 halide or Fe + 2 halide complexes with triphenylphosphine, indanyl or cyclopentadienyl).

In some embodiments, an approximately stoichiometric amount of metal compound and ligand is used in the catalyst, based on the molar ratios of the components when the metal ion is completely complexed. In other embodiments, the relationship between the metal compound and the ligand is in the

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interval 1: (0.5 to 2) or in the interval 1: (0.8: 1.25).

Generally, when the catalyst is copper, bidentate or multidentate nitrogen ligands produce more active catalysts. In addition, bridged or cyclic ligands and branched aliphatic polyamines provide more active catalysts than simple linear ligands. When bromine is the counterion, bidentate or tetradentate ligands for Cu + 1 are needed. When more complex counterions such as triflate or hexafluorophosphate are used, two bidentate or one tetradentate ligands can be used. The addition of metallic copper may be advantageous in some embodiments, particularly when faster polymerization is desired, since metallic copper and Cu + 2 can undergo a redox reaction to form Cu + 1. The addition of part of Cu + 2 at the beginning of some ATRP reactions can be used to decrease the amount of normal termination.

In some embodiments, the amount of catalyst employed in the polymerization reactions is the molar equivalent of the initiator that is present. Since the catalyst is not consumed in the reaction, however, it is not essential to include an amount of catalyst as high as that of the initiator. The ratio of catalyst to the initiator, based on the transition metal compound in some embodiments is about 1: (1 to 50), and in other embodiments about 1: (1 to 10).

E.3.1.3 Polymerization conditions

In some embodiments, the live radical polymerization process of the invention is preferably performed to achieve a degree of polymerization in the range of 3 to about 2000, and in other embodiments of about 5 to about 500. The degree of polymerization in other embodiments they are in the range of 10 to 100, or alternatively, in the range of about 10 to about 50. The degree of polymerization in the group or atom transfer radical polymerization technique is directly related to the initial initiator ratio with respect to monomer. Therefore, in some embodiments, the initial initiator ratios with respect to monomer are in the range of 1: (3 to about 2,000) or about 1: (5 to 500), or about 1: (10 to 100), or about 1: (10 to 50).

Polymerization reactions are typically carried out in the liquid phase, using a single homogeneous solution. However, the reaction can be heterogeneous, comprising a solid and a liquid phase (for example, an aqueous suspension or emulsion). In those embodiments in which a non-polymerizable solvent is used, the solvent employed is selected taking into account the nature of the zwitterionic monomer, the initiator, the catalyst and its ligand; and also, any comonomer that can be used.

The solvent may comprise a single compound or a mixture of compounds. In some embodiments, the solvent is water and, in other embodiments, water is present in an amount of about 10% to about 100% by weight, based on the weight of the monomers present in the reaction. In those embodiments in which a water insoluble comonomer is to be polymerized with a zwitterionic monomer, it may be desirable to employ a solvent or co-solvent (together with water) that allows solubilization of all monomers present. Suitable organic solvents include, without limitation, formamides (for example, dimethylformamide), ethers (for example, tetrahydrofuran), esters (ethyl acetate) and, much more preferably, alcohols. In some embodiments in which a mixture of water and organic solvent is to be used, the C1-C4 alkyl alcohols miscible with water (methanol, propanol ethanol, isopropanol, butanol, isobutanol and tercbutanol) are useful organic solvents. In other embodiments, the combinations of water and methanol are suitable for polymerization reactions. The reaction can also be performed in supercritical solvents such as CO2.

As indicated above, in some embodiments, it is desirable to include water in the polymerization mixture in an amount of about 10% to about 100% by weight based on the weight of the monomers to be polymerized. In other embodiments, the total non-polymerizable solvent is from about 1% to about 500% by weight, based on the weight of the monomers present in the reaction mixture. In other embodiments, In other embodiments, the total non-polymerizable solvent is from about 10% to about 500% by weight or, alternatively, from 20% to 400%, based on the weight of the monomers present in the mixture of reaction.

In some embodiments, the contact time of the zwitterionic monomer and the water before contact with the initiator and the catalyst is minimized by forming a premix comprising all components other than the zwitterionic monomer and for the zwitterionic monomer to be added to the premix last. place.

Polymerization reactions can be performed at any suitable temperature. In some embodiments, the temperature may be from about room temperature (room temperature) to about 120 ° C. In other embodiments, the polymerizations can be carried out at an elevated temperature from room temperature in the range of about 60 ° to 80 ° C. In other embodiments, the reaction is performed at room temperature.

In some embodiments, the compounds of the invention have a polydispersity (of molecular weight) of less

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1.5, as judged by gel permeation chromatography. In other embodiments, the polydispersities may be in the range of 1.2 to 1.4. The polydispersity can be reduced by processing the post-polymerization reaction of the compounds of the formulas (Ia) to (Vb) (for example, size selection in chromatographic gel permeation media). Alternatively, when the coupling of the compounds of the formulas (Ia), (I Ia), (11 Ia), (IVa), or (Va) to a biologically active agent is to be performed to prepare the desired conjugates, having The intermediate compounds formulas (Ia), (I Ia), (II Ia), (IVa) or (Va) can be selected by size (for example, by chromatography) before coupling to the biologically active agent.

The polymer residues present in the compounds of the invention, particularly those prepared by living radical polymerization, can be used to form block copolymers where the product of the first polymerization is used as an initiator in a second group or radical polymerization stage of atom transfer performed in the presence of a catalyst, and one or more additional ethylenically unsaturated monomers. The product is a block copolymer of type AB, where the second block "B" can be composed of monomers of a different type from those used in the initial block A. Alternatively, the monomers from which the block is formed A and block B comprise the same component monomers present in different relationships. More often they comprise different monomers, although they may include common comonomers. Additional blocks may be added to produce linear block copolymers having the sequence ABC or ABA. The ABA type block copolymer can also be produced using a difunctional initiator to which type B monomers can be added. In the second polymerization, the monomer blocks A will be added to the two ends of the B polymers that act as an initiator that produces a polymer of the ABA type. Similarly, block copolymers having star or comb architectures can be generated from multifunctional initiators used to prepare the star or comb polymers described above. When necessary, a different catalyst or solvent system can be used to prepare the second or subsequent polymer blocks.

E.3.1.4 Preparation of compounds having a non-halogenated polymer end

In those embodiments of compounds of the formulas (Ia) to (Vb), in which a halogen is attached to the end of the polymeric group due to the use of a halogen-containing initiator or catalyst in the polymerization reaction, it may be desirable to replace the Halogen with other functionality. A variety of reactions can be used for the conversion of the aliphatic halogen. In one embodiment, the aliphatic halogen conversion may comprise an aliphatic nucleophilic substitution reaction. In one embodiment, the aliphatic nucleophilic reaction is an deologenation of isocyanate, and in another embodiment an dehalogenation of isothiocyanate. The resulting isocyanates or isothiocyanates can be converted into the corresponding amine by treatment with acid or base. Since isothiocyanates can give the alkylation of S, and tend to require more vigorous conditions to convert them into the corresponding amine, isocyanates are preferred over isothiocyanates in dehalogenation reactions. When the aliphatic halide is treated with isocyanate in the presence of an alcohol, carbamates can be prepared directly.

A halogen attached to the end of the polymeric group may also be subjected to nucleophilic substitution by a suitable nucleophile and become an alcohol, an alkyl ether (for example, -O-methyl or -O-ethyl), an ester, a thioether and the like. . Halogens may also be subject to an elimination reaction to give rise to an alkene (double bond).

To avoid affecting a biological agent coupled to a compound of the formulas (Ib), (11 b), (IIIb), (IVb), or (Vb), the conversion of terminal aliphatic halogens can be carried out in compounds of formula (Ia ), (I Ia), (II Ia), (IVa) or (Va) before coupling to a biologically active agent.

E.3.2 Coupling of biologically active agents to compounds of the formulas (Ia), (IIa), (IIIa), (IVa), or (Va).

The preparation of the compounds (Ib), (IIb), (IIIb), (IVb), or (Vb), which comprise a biologically active agent, can be performed by first attaching the biologically active agent to a linking group and subjecting the agent biologically active coupled to conditions suitable for the synthesis of the polymer group of the molecule. In these cases, a suitable linking group can be an initiator (eg, iodinated, brominated or chlorinated compound / group) for use in ATRP reactions. Such a reaction scheme is possible when the biologically active agent is compatible with the polymerization reactions of the polymer and any subsequent processing required. However, the coupling of biologically active agents to compounds of formula (Ia), (IIa), (IIIa), (IVa), or (Va), can be advantageously used to prepare compounds of the formulas (Ib), (IIb ), (IIIb), (IVb), or (Vb), particularly when the biologically active agent is not compatible with the conditions suitable for polymerization. Furthermore, when the cost causes the loss of an agent to produce imperfect synthetic yields, particularly in multi-stage synthetic reactions, the coupling of the biologically active agent to the compounds of the formulas (Ia), (IIa), (IIIa), ( IVa) or (Va) can be used advantageously.

The compounds of the formulas (Ib), (IIb), (IIIb), (IVb) or (Vb) can be prepared by reaction with a compound of

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formula (la), (Ila), (Illa), (IVa), or (Va) with a biologically active agent. In those cases in which the compounds of the formulas (Ia), (IIa), (IIIa), (IVa) or (Va) carry a group - (Sp1) nX (see, for example, Scheme I, part IA and Scheme II, part IIA), where group X is reactive, the compounds can react directly with a biologically active agent to prepare a molecule of the formulas (Ib), (IIb), (IIIb), (IVb) or (Vb) . When the X group is present in a protected form, the molecule can be subjected to deprotection before the reaction with a biologically active agent (for example, protected carboxylic acids, hydroxyls, amines, carbonyls and the like from which the X groups can be deprotected) .

When a group - (Sp1) n-X is not compatible with the conditions used for the polymerization reactions, it may be desirable to introduce the group after the polymerization reaction (see, for example, Scheme I part IB and Scheme II part IIB). Such a situation may arise when a group X contains a double bond that cannot be present in the polymerization reaction (for example, X comprises a vinylpyridine group, an unsaturated alpha-beta carbonyl, an alpha-beta ester, an alpha-beta amide unsaturated, or a maleimide group). In such circumstances, an initiator carrying one or more halogens can provide a group L with suitable functionalities (for example, hydroxyl, amine or carboxyl) for the attachment of moieties - (Sp1) n-X. In one such embodiment, a thio-reactive group of malemide and a linker comprising an alkyl group terminated in a carboxyl can be attached to a group L which carries an aldehyde after polymerization by reaction with N- (p-maleimidopropionic acid ) hydrazide-TFA (BMPH, available from Pierce Biotechnology).

As discussed above, the biologically active agent A, of a compound of formula (Ib), (IIb), (IIIb), (IVb), or (Vb) may include a spacer group, represented as (Sp2) p, where p is 0 or 1, attached thereto as a means to bind the biologically active agent. Said binding groups can be introduced to provide not only a spacer group, but also a group capable of reacting with a group X of a compound of the formulas (Ia), (IIa), (IIIa), (IVa), or (Va )).

The introduction of the Sp2 spacer groups into biologically active molecules can occur before the reaction of the biologically active agent with a compound of the formulas (Ia), (IIa), (IIIa), (IVa), or (Va). The incorporation of Sp2 spacers into biologically active agents is particularly desirable when they can be used to introduce a unique reactive functionality that can react with a group X to form the group Z of a compound of the formulas (Ib), (IIb), (IIIb) , (IVb), or (Vb).

When desirable and chemistry allows, Sp2 binding groups can be linked to the group X of a compound of the formulas (Ia), (IIa), (IIIa), (IVa) or (Va) before a reaction with the biologically active agent. Said synthetic strategy can be used in those embodiments in which, for example, the biologically active agent is expensive, or has a limited life due to its susceptibility to degradation. In these cases, it is desirable that the final coupling between a Sp spacer group that has been linked to an X functionality of the compounds (Ia), (IIa), (IIIa), (IVa), or (Va) (ie, to form a compound having -X converted to -Z-Sp2) is compatible with other functionalities present in the molecules, including phosphorylcholine groups that are present in the polymer.

The coupling of biologically active agents to the compounds of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) can be performed using chemical conditions and reagents applicable to the reactions that are performed. When, for example, the coupling requires the formation of an ester or an amide, the dehydration reactions between a carboxylic acid and an alcohol or amine can employ a dehydrating agent (for example, a carbodiimide such as dicyclohexylcarbodimide, DCC, or the agent Water soluble 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide hydrochloride, EDC). Alternatively, esters of N-hydroxysuccinimide (NHS) can be used to prepare amides. The reaction to prepare amides using NHS esters is typically performed at almost neutral pH in phosphate, bicarbonate, borate, HEPES or other non-amine-containing buffers from 4 ° C to 25 ° C. In some embodiments, reactions using EDC as a dehydrating agent, a pH of 4.5-7.5 can be used; In other embodiments, a pH of 4.5 to 5 may be employed. Morpholinoethanesulfonic acid, MES, is an effective carbodiimide reaction buffer.

Thiol groups can be reacted under a variety of conditions to prepare different products. When a thiol is reacted with a maleimide to form a thioether bond, the reaction is typically performed at a pH of 6.5-7.5. Excess maleimide groups can be inactivated by the addition of free thiol reagents such as mercaptoethanol. When the disulfide bonds are present as a linkage, they can be prepared by thiol-disulfide exchange between a sulfhydryl present in the biologically active group and an X functionality that is a disulfide such as a pyridyl disulfide. The reactions involving pyridyl disulfides can be carried out at pH 4-pH 5 and the reaction can be monitored at 343 nm to detect the released pyridin-2-thione. Thiol groups can also be reacted with epoxides in an aqueous solution to produce hydroxy thioethers.

The reaction of guanido groups (for example, those of an arginine in a protein or polypeptide of interest) with a glyoxal can be carried out at pH 7.0-8.0. The reaction typically proceeds at 25 ° C. The derivative, which contains two phenylglyoxal residues per guanide group, is more stable under slightly acidic conditions (below pH 4) than at neutral or alkaline pH, and allows the isolation of bound materials. At neutral or alkaline pH values, the junction is

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decomposes slowly. When an arginine residue of a protein or polypeptide is reacted with a phenylglyoxal reagent, approximately 80% of the bond will be hydrolyzed to regenerate the original arginine residue (in the absence of excess reagent) in approximately 48 hours at 37 ° C at about pH 7.

Imidoester reactions with amines are typically performed at a pH of 8-10, and preferably at about pH 10. The amidine bond formed from the reaction of an imidoester with an amine is reversible, particularly at high pH.

Haloacetals can be reacted with sulfhydryl groups over a wide pH range. To avoid secondary reactions between histidine residues that may be present, particularly when the sulfhydryl group is present in a protein or polypeptide, the reaction can be carried out at approximately pH 8.3.

Aldehydes can react with amines under a variety of conditions to form imines. When the aldehyde or the amine are immediately adjacent to an aryl group, the product is a Schiff base that tends to be more stable than when an aryl group is not present. Conditions for the reaction of amines with aldehydes to form an imine bond include the use of a basic pH of about pH 9 at about pH 11 and a temperature of about 0 ° C at room temperature, for 1 to 24 hours. Buffers that include buffers containing borohydride and tertiary amines are often used for the preparation of imines. When desired, imine conjugates, which are hydrolytically susceptible, can be reduced to form an amine bond that is not hydrolytically susceptible. The reduction can be performed with a variety of suitable reducing agents that include sodium borohydride or sodium cyanoborohydride.

The reaction conditions provided above are intended to provide general guidance to the expert. The person skilled in the art will recognize that the reaction conditions can be varied as necessary to promote the formation of conjugates of the formulas (Ib), (Ilb), (IlIb), (IVb) or (Vb) and that orientation for modification of the reactions can be obtained from standard texts in organic chemistry. Additional guidance can be obtained from texts such as Wong, S.S., "Chemistry of Protein Conjugation and Cross-Linking," (CRC Press 1991), which analyze related chemical reactions.

E.4 Alternatives to live radical polymerization methods for the preparation of compounds of the invention

In addition to the use of the live radical polymerization methods described above, the compounds of the present invention can also be prepared by various methods including the preparation of the polyacrylic acid polymer portions of the compounds to form polymeric intermediates through any method. known in the art. Suitable methods include free radical polymerization methods that can be initiated by methods that include thermal, hydrolytic, redox or photochemical means. Polymeric intermediates can be selected by size by chromatographic methods, if necessary, and coupled to a reactive group (i.e., a group X) through any group capable of providing a group L (and a Sp1 spacer if present) that it carries a reactive group, or a protected form thereof, to form a compound of formula (Ia) (IIa), (IIIa), (IVa), or (Va). After any necessary deprotection or activation of group X, the compounds of the formulas (Ib) (11 b), (IIIb), (IVb), or (Vb) can be prepared by reaction with a biologically active agent under suitable conditions. This method of preparation is illustrated in Scheme III for the "M" monomers and 4-formylbenzoic acid, which provides a means (the carboxyl group) for coupling the intermediate polymer and a reactive group (the aldehyde) for reaction with an agent. biologically active that carries amine.

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image44

Scheme III

M (monomer)

polymerization

HO- (polyM) -OH

+ agent

reducer, for example, Fe * 4.

0 2, v 2, Cu z

FCMC

etc (initiator)

4-formylbenzoic acid

(where P represents an aldehyde protecting group)

dcshidratacion reaction with

a carbodnmide, etc.

protection

POHC

OHC

(polyM) -OH

O— (polyM) -OH

A-NHi

Biologically active agent

Grassroots training

wearing an amine

from Schiff

A-N = HC

(polyM) -OH

The imine product prepared in Scheme III can be reduced to produce an amine by the use of suitable reducing agents such as sodium cyanoborohydride.

E.5 Purification and determination of molecular weight.

When desirable, the compounds of formulas (Ia) to (Vb) can be purified by any known means, including precipitation, dialysis and chromatography. A convenient means to remove contaminants from small molecules such as catalysts, ligands, non-polymerized monomers, or small-molecule active biological agents from the desired polymeric materials is the passage of the product on a desalted gel column balanced with a suitable buffer ( for example, Bio-Gel P6DG available from Bio-Rad Laboratories, Inc., Hercules CA, which can be used with conjugates of more than 6,000 Daltons).

Chromatography can also be performed on media capable of resolving materials having a molecular weight in the range of polymeric and polymer conjugated materials of formulas (Ia) to (Vb), particularly when it is desirable to determine the polydispersity of the conjugated polymer. Any conventional chromatographic medium capable of resolving materials in the desired molecular weight range including Superdex, Sephacryl, Sephadex and Sepharose can be employed. The specific choice of media (for example, Superdex peptide, Superdex 75 or Superdex 200) will be affected by the size of the conjugate to be resolved. Chromatography can be performed using standard column chromatographic methods, fast protein liquid chromatography (FPLC), or HPLC. Although chromatography at room temperature is possible in many cases, chromatography at lower temperatures (for example, 4 ° C) is often desirable. Any suitable buffer can be used in the chromatographic separation of the polymer conjugates. Chromatography is also possible in a mobile phase which is also a pharmaceutically acceptable excipient composition, including, but not limited to, normal saline (0.9 g of NaCl per 100 ml of water) or phosphate buffered saline (PBS) . The use of pharmaceutically acceptable excipients as a mobile phase has the advantage of avoiding subsequent manipulations to exchange buffers present with polymeric materials.

Other forms of chromatography may also be employed, including, but not limited to, ion exchange chromatography and hydrophobic interaction chromatography.

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The present invention is further illustrated by the following examples.

Examples

Example 1. Preparation of 2-ethylacrylic acid from diethyl malonate.

Ethylacrylic acid was prepared from diethyl malonate by the preparation of 2-carbetoxybutyric acid which was subsequently converted to 2-ethylacrylic acid (EAA). 2-carboethoxybutyric acid.

Diethyl ethylmalonate was stirred overnight with 95% ethanol in the presence of 1M KOH. The white precipitate that formed was removed by filtration of the solution, and the filtrate was concentrated under reduced pressure (e.g., in a rotary evaporator). The yellowish oil obtained after concentration of the filtrate was added to the precipitate and the mixture was dissolved in a minimum amount of water. After acidification with dilute aqueous HCl at a pH of 2, an oil is separated from the solution. The oil was taken up in diethyl ether and the aqueous layer was extracted three more times with diethyl ether. The ether extracts were combined, dried over magnesium sulfate and filtered. A quantitative yield of 2-carboethoxybutyric acid was obtained in the form of a yellowish oil.

2-ethylacrylic acid (ethyl 2-ethyl acrylate).

The 2-carboethoxybutyric acid obtained in step (a) was cooled to -5 ° C and diethylamine was added. A solution of formalin was added dropwise to the cooled reaction mixture which was then allowed to warm to room temperature. After stirring for 24 h, the reaction mixture was heated to 60 ° C and stirred for a further 8 h. The mixture consisted of two layers, which were cooled to 0 ° C. Concentrated sulfuric acid was added and the mixture was extracted with three 200 ml portions of diethyl ether. The ether extracts were combined, dried over magnesium sulfate and filtered. The ether was removed under reduced pressure to produce ethyl 2-ethyl acrylate as a yellow oil.

Example 2. Preparation of 2-methacryloxyethyl phosphorylcholine (HEMA-PC or MPC).

image45

To a solution of 2-hydroxyethyl methacrylate (HEMA) (4.4 g, 40 mmol) in dry THF (40 ml) was added dry triethylamine (3.4 g, 40 mmol). This mixture was stirred at room temperature for 3 minutes, then cooled to -20 ° C. To the cooled solution was added COP (4.5 g, 40 mmol) in the form of a solution in THF (20 ml) over a period of 1 hour. The temperature of the reaction mixture was maintained at approximately -20 ° C. The white precipitate was filtered, and the remaining solution was concentrated under reduced pressure to yield 6.7 g (71%) of 2- (2-oxo-1,3, 2, dioxaphospholoyloxy) ethyl methacrylate (OPEMA) in form of a pale yellow liquid. OPEMA (4.0 g, 16 mmol) in dry acetonitrile (30 ml) was stirred in a reaction bottle. The bottle was cooled to -20 ° C, and anhydrous trimethylamine (4 ml) was quickly added to the solution. The bottle was quickly closed, and the mixture was heated at 60 ° C for 24 hours, then kept at -10 ° C for 12 hours. MPC was observed as a white precipitate, collected by filtration, washed with cold acetonitrile, and dried under reduced pressure to give 1.5 g of product as a white powder. 1H NMR (300 MHz, CDCh): 5.93 (s, 3H), 3.40 (s, 9H), 3.80-3.85 (m, 2H), 4.09-4.11 (m , 2H), 4.30-4.36 (m, 4H), 5.84 (s, 1H), 6.11 (s, 1H) ppm.

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Example 3. Preparation of pyrrolidin-1-yl ester of 2-bromo-2-methyl-propionic acid.

image46

A solution of W-hydroxysuccinimide (NHS) (3.0 g, 26 mmol) in dry methylene chloride (300 ml) was stirred under an inert atmosphere (argon or nitrogen). Triethylamine (7.3 ml, 52 mmol) was added, the mixture was cooled to 0 ° C, and 2-bromo-2-methylpropionyl bromide (3.9 ml, 31 mmol) was added dropwise over a period of 15 minutes. The mixture was stirred for 1 hour at 0 ° C, then heated to room temperature and stirred overnight. Most of the solvent was removed by evaporation, and the remaining mixture was diluted with cold water (400 ml), and extracted with diethyl ether (3 x 30 ml), then aqueous potassium carbonate (3 x 30 ml) was washed and 1M HCl (aq.) (3 x 30 ml). The organic layer was dried over anhydrous magnesium sulfate, then filtered and concentrated to give a brown solid. Crystallization of ethyl acetate gave the desired product as a white powder (2.62 g, 39%). 1 H NMR (CDCla) or (ppm) 2.09 (s, 6H, CHa ^ Br), 2.88 (s, 4H, Hnhs); 13C NMR (CDCl3) or (ppm) 25.7 (Cnhs), 30.8 (C (CH3) 2Br), 51.3 (C (CH3) 2Br), 167.6 (C = O), 168.7 (Cnhs = O).

Example 4. Representative polymerization of MPC using 2-bromo-2-methyl-propionic acid pyrrolidin-1-yl ester of Example 3 and atom transfer radical polymerization (ATRP).

image47

2-Bromo-2-methyl-propionic acid pyrrolidin-1-yl ester of Example 3 (13 mg, 0.050 mmol) was dissolved in degassed DMSO (1.5 ml). To this solution was added Cu1Br (7 mg, 0.05) mmol) and 2,2'-bipyridine (15 mg, 0.10 mmol), followed by a solution of MPC (547 mg, 1.85 mmol) in degassed methanol (0.5 ml). The reaction mixture was subjected to three freeze-pump-thaw cycles, then stirred in an inert atmosphere (argon or nitrogen) at room temperature for 18 hours, then at 40 ° C for 4 hours. The mixture was passed through a short column of silica gel with methanol as eluent, to give a colorless solution. The solution was dried under vacuum to give the desired polymer (190 mg) as a white powder. 1H NMR (400 MHz, CD3OD): or 0.81-1.33 (a, 35H), 1.81-2.22 (a, 22H), 2.87 (s, 4H), 3.25 (s , 132H), 3.69-3.81 (a, 19H), 4.04-4.45 (a, 60H) ppm; 31P NMR (400 MHz, CD3OD): -0.34 ppm. Gel permeation chromatography, against calibration standards of poly (ethylene oxide), eluting with 0.1 M aqueous NaNO3 containing 0.2 percent NaN3 by weight: Mn 8,900, Mp 9,600, Mw 11,600, PDI 1, 30 The following table provides examples of this polymerization performed under a variety of conditions.

MPC polymers terminated in NHS prepared under different conditions.

 Sample number

 Theoretical DP solvent Mn calc. DP (NMR) Mn (NMR) Mn (GPC) PDI (GPC)

 I

 MeOH NHS-MPC10 2960 NHS-MPC8 2,400 3,500 1.15

 II

 DMSO NHS-MPC20 5920 NHS-MPC20 5.00 11,000 2.4

 III

 MeOH-DMSO (1: 1) NHS-MPC25 7400 NHS-MPC20 5,900 7,700 1.15

 IV

 MeOH-DMSO (1: 1) NHS-MPC25 7400 NHS-MPC15 4,400 5,400 1.25

 V

 MeOH-DMSO (1: 3) NHS-MPC37 10989 NHS-MPC28 8,200 9,600 1.30

 DP = degree of polymerization (n monomeric repeating units); Mn = weight average molecular weight; GPC = gel permeation chromatography; PDI = polydispersity index, as defined by Mw / Mn, or weight average molecular weight divided by number average molecular weight (both determined by GPC).

Example 5. Preparation of 4-formyl-phenyl ester of 2-bromo-2-methyl-propionic acid.

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A solution of 4-hydroxy benzaldehyde (6.1 g, 0.050 mol) and triethylamine (6.1 g, 0.060 mol) was stirred in THF (200 ml) in a round bottom flask. Bromoisobutyryl bromide (13.6 g, 0.060 mol) was added slowly to the benzaldehyde solution under stirring, and the reaction mixture was stirred at room temperature for five hours. The white precipitate that appeared was removed by filtration, and the solvent was removed by rotary evaporation. Purification by column chromatography (eluting with 12% EtOAc in hexane) provided the desired aldehyde (6.5 g) as a white solid. 1 H NMR (CDCh) or = 2.09 (s, 6H), 7.33 (d, J = 7.2 Hz, 2H), 7.96 (d, J = 7.99 Hz, 2H), 10, 02 (s, 1 H) ppm; 13C NMR (CDCla) or (ppm) 30.4, 55.3, 121.5, 131.3, 134.2, 155.3, 169.6, 175.7, 190.9 ppm.

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Example 6. Polymerization of MPC using benzaldehyde initiator and ATRP.

image49

The 2-bromo-2-methylpropionic acid 4-formyl-phenyl ester of Example 5 (10.8 mg, 40.0 mmol) was dissolved in degassed DMSO (1.0 ml). To this solution were added CuBr (7 mg, 0.05 mmol), 2,2'-bipyridine (15 mg, 0.10 mmol), and a solution of MPC (296 mg, 1.00 mmol) in degassed methanol (0.5 ml). The reaction mixture was subjected to three freeze-pump-thaw cycles, then stirred under a nitrogen atmosphere for 18

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hours at room temperature, and 4 hours at 40 ° C. The mixture was passed through a short column of silica gel, eluting with methanol, and washed with dry THF (2 ml) to give a colorless solution. The solution was dried under vacuum to give the desired polymer (100 mg) as a white powder. 1H NMR (300 MHz, CD3OD): or = 0.611.42 (m, 236H), 1.90-2.33 (m, 131H),), 3.32 (s, 766H), 3.69-3, 81 (sa, 140H), 3.95-4.43 (m, 438H), 7.27-7.46 (sa, 2H), 7.93-8.12 (sa, 2H), 9.95- 10.11 (sa, 1 H); GPC (0.1 M aqueous NaNOa eluent containing 0.2% NaNa by weight and PEO as standard): Mn: 8.300; Mp: 9,000; Mw: 11,000; POI 1.29.

Example 7. Preparation of 3- (2-Bromo-2-methyl-propionyloxy) -5-formyl-phenyl ester of 2-bromo-2-methyl-propionic acid.

image50

A solution of 3,5-dihydroxybenzaldehyde (1.38 g, 10.0 mmol) and triethylamine (2.5 g, 25 mmol) in dry THF (40 ml) was stirred at room temperature. Bromoisobutyryl bromide (5.7 g, 25 mmol) was added slowly to the stirring mixture, and the reaction was allowed to proceed at room temperature for 12 hours. A white precipitate formed, which was removed by filtration. The solvent was removed by rotary evaporation, and the desired compound was purified by column chromatography eluting with 15% EtOAc / hexanes to give 1.7 g (40%) of the desired difunctional initiator as a pale yellow viscous liquid. . 1H NMR (300 MHz, CDCh): or = 2.06 (s, 12H), 7.31 (t, J = 2.5 Hz, 1H), 7.61 (d, J = 2.5 Hz, 2H ), 10.05 (s, 1H), 13C NMR (400 MHz, CDCh): or = 30.2, 54.7, 119.8, 120.9, 138.2, 151.2, 169.5, 189.8.

Example 8. MPC polymerization using branched benzaldehyde initiator and ATRP.

image51

2-Bromo-2-methyl-propionic acid 3- (2-bromo-2-methyl-propionyloxy) -5-formylphenyl ester (25 mg, 0.060 mmol) of Example 7 was dissolved in degassed DMSO (1.5 ml) . To this solution were added CuBr (14.3 mg, 0.100) mmol), 2,2'-bipyridine (30 mg, 0.20 mmol), and a solution of MPC (444 mg, 1.50 mmol) in methanol degassed (0.5 ml). The reaction mixture was subjected to three freeze-pump-thaw cycles, then stirred under a nitrogen atmosphere for 18 hours at room temperature, and 4 hours at 40 ° C. The mixture was passed through a short column of silica gel, eluting with methanol, to give a colorless solution. The solution was dried in vacuo and washed with dry THF (2 ml) to give the desired polymer (250 mg) as a white powder. 1H NMR (400 MHz, CD3OD): or 0.52-1.54 (m, 254H), 1.85-2.49 (m, 143H),), 3.35 (s, 790H), 3.65 -3.79 (sa, 148H), 4.154.43 (m, 474H), 7.27-7.46 (sa, 2H), 7.6-7.7 (sa, 1H), 9.90-10 , 1 (1H); GPC (polyethylene oxide standards, and elution with 0.1 M aqueous NaNO3 containing 0.2% NaN3 by weight: Mn: 9100; Mp: 10,000; Mw: 11,500; PDI 1.3.

Example 9. Preparation of 2- (2,2-dimethoxy-ethoxy) -ethanol.

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A solution of potassium hydroxide (5.0 g, 89 mmol) in ethylene glycol (12.5 ml, 224 mmol) was stirred at 115 ° C in a round bottom flask. After the solution of potassium hydroxide, chloroacetaldehyde dimethyl acetal (8.0 ml, 44 mmol) was added dropwise over 15 minutes, and the reaction mixture was stirred for 66 hours. Then, the mixture was allowed to cool to room temperature, and the whole was diluted with water (40 ml), and then extracted with methylene chloride (3 x 20 ml). The organic layer was washed with brine (3 x 20 ml), dried over magnesium sulfate, then concentrated to give the desired dimethyl acetal product as a very pale yellow liquid (2.2 g, 45% ). 1H NMR (300 MHz, CDCls): or = 2.03 (s), 2.98 (s, 1H), 3.31 (s, 6H), 3.48 (t, 2H), 3.52 (t , 2H), 3.68 (t, 2H), 4.48 (c, 1H) 13C NMR (300 MHz, CDCh): or = 53.9, 61.5, 70.5, 72.9, 102, 5.

Example 10. 2- (2,2-Dimethoxy-ethoxy) -ethyl ester of 2-bromo-2-methyl-propionic acid.

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A solution of 2- (2,2-dimethoxy-ethoxy) -ethanol (2.20 g, 14.6 mmol) of Example 9 in dry triethylamine (3.0 ml, 21 mmol) and dry methylene chloride (30 ml ) stirred at 0 ° C. Then, 2-bromo-2-methylpropionyl bromide (1.7 ml, 14 mmol) methylene chloride (10 ml) was added to the alcohol over a period of 15 minutes. The reaction mixture was heated to room temperature and stirred overnight. The reaction mixture was filtered, the filtrate was washed with saturated sodium bicarbonate, and dried with magnesium sulfate. The solvent was removed and the yellow oil was purified by column chromatography. 1H NMR (300 MHz, CDCla): or = 1.78 (s, 6H), 3.31 (s, 6H), 3.49 (d, 2H), 3.68 (t, 2H), 4.25 (t, 2H), 4.46 (t, 1H) 13C NMR (300 MHz, CDCla): or = 31.23, 55.85, 56.15, 65.58, 69.55, 71.44, 103 , 09, 172.07.

Example 11. MPC polymerization using protected aliphatic aldehyde initiator and ATRP.

or

image54

OR

/

o = p — oe i

or

X

®NMe3

2- (2,2-Dimethoxy-ethoxy) -ethyl ester of 2-bromo-2-methyl-propionic acid (16 mg, 0.080 mmol) of Example 10 was dissolved in degassed DMSO (2 ml). To this solution was added CuBr (12.5 mg, 0.088 mmol) and 2,2'-bipyridine (27 mg, 0.17 mmol), followed by a solution of MPC (592 mg, 2.00 mmol) in methanol degassed (0.5 ml). The reaction mixture was subjected to three freeze-pump-thaw cycles, then stirred under a nitrogen atmosphere for 18 hours at room temperature, and 4 hours at 40 ° C. The mixture was passed through a short column of silica gel, eluting with methanol, to give a colorless solution. The solution was dried in vacuo and washed with dry THF (2 ml) to give the desired polymer (350 mg) as a white powder. GPC (polyethylene oxide standards, and elution with 0.1 M aqueous NaNO3 containing 0.2% NaN by weight). 1 H NMR (400 MHz, CD3OD): or 0.81-1.33 (a), 1.70-2.22 (a), 3.31-3.49 (a), 3.67-3.84 (a), 4.04-4.45 (a) ppm; Mn: 11,200; Mp: 11,300; Mw: 15,600; POI 1.4.

Example 12. Conjugation of the aldehyde functionalized polymer of Example 6 with G-CSF.

The aldehyde functionalized polymer of Example 6 (6.3 mg) was dissolved in 40 mM sodium cyanoborohydride (2 ml) prepared in 100 mM sodium acetate buffer at pH 4.6. Granulocyte colony stimulating factor (G-CSF, 0.5 ml, figure 2) purified from a transgenic chicken (for example, as described in US Patent No. 6,730,822) was added in Formulation buffer at a concentration of 0.265 mg / ml to 1.5 ml of the polymer and stirred at room temperature for 24 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (Figure 3 (1: conjugate; 2: G-CSF; 3: buffer)).

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Example 13. Alternative conjugation of the aldehyde functionalized polymer of Example 6 with G-CSF.

The aldehyde functionalized polymer of Example 6 (6.3 mg) was dissolved in 40 mM sodium cyanoborohydride (2 ml) prepared in 100 mM sodium acetate buffer at pH 4.6. The granulocyte colony stimulating factor (G-CSF, 0.1 ml, figure 2) obtained from a transgenic chicken (for example, as described in US Patent No. 6,730,822) in buffer formulation at a concentration of 0.265 mg / ml was added to 0.5 ml of the polymer and stirred at room temperature for 48 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (Figure 4 (4: conjugate; 5: G-CSF; 6: buffer)).

Example 14. Conjugation of the aldehyde functionalized polymer of Example 6 with EPO.

The aldehyde functionalized polymer of Example 6 (2.5 mg) was dissolved in 80 mM sodium cyanoborohydride (1 ml) prepared in 100 mM sodium acetate buffer at pH 4.6. Erythropoietin (EPO), 0.1 ml, figure 1) obtained from a transgenic chicken (for example, as described in US Patent No. 6,730,822) in 100 mM sodium phosphate buffer (pH 6 , 98) at a concentration of 0.44 mg / ml was added to 0.5 ml of the polymer and stirred at room temperature for 72 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (after 24 hours, figure 5 (7: conjugate; 8: EPO; 9: buffer); after 48 hours, Figure 6 (10: conjugate; 11: EPO; 12: buffer); and after 72 hours, Figure 7 (13: conjugate; 14: EPO; 15: buffer)).

Example 15. Conjugation of the NHS functionalized polymer of Example 4 with G-CSF.

The NHS functionalized polymer of Example 4 (Sample I) (1.9 mg) was dissolved in G-CSF (0.1 ml, Figure 2) obtained from a transgenic chicken (for example, as described in the Patent of United States No.

6,730,822) in 100 mM sodium borate buffer (pH 9.3) at a concentration of 0.265 mg / ml and stirred at room temperature for 24 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (Figure 8 (16: conjugate; 17: G-CSF; 18: buffer)).

Example 16. Conjugation of the NHS functionalized polymer of Example 4 (Sample I) with EPO.

The NHS functionalized polymer of Example 4 (Sample I) (2.1 mg) was dissolved in EPO (0.2 ml, figure 1) obtained from a transgenic chicken (for example, as described in US Pat. No. 6,730,822) in 100 mM sodium phosphate buffer (pH 8) at a concentration of 0.44 mg / ml and stirred at room temperature for 72 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (after 24 hours, figure 9 (19: conjugate; 20: EPO; 21: buffer); and after 72 hours, Figure 10 (22: conjugate; 23: EPO; 24: buffer)).

Example 17. Conjugation of the NHS functionalized polymer of Example 4 with interferon alfa.

The NHS functionalized polymer of Example 4 (Sample I) (1.8 mg) was dissolved in interferon alfa2b (0.2 ml) obtained from a transgenic chicken (for example, as described in US Pat. No. . °

6,730,822) in 100 mM sodium borate buffer (pH 9.3) at a concentration of 0.035 mg / ml and stirred at room temperature for 24 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (Figure 11 (25: conjugate; 26: alpha interferon; 27: buffer)).

Example 18. Alternative conjugation of the NHS functionalized polymer of Example 4 with G-CSF.

The NHS functionalized polymer of Example 4 (Sample V) (1.7 mg) was dissolved in G-CSF (0.3 ml, Figure 2) obtained from a transgenic chicken (for example, as described in the Patent of United States No.

6,730,822) in 100 mM sodium borate buffer (pH 9.3) at a concentration of 0.265 mg / ml and stirred at room temperature for 72 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (Figure 12 (28: conjugate; 29: G-CSF; 30: buffer)).

Example 19. Conjugation of the NHS functionalized polymer of Example 4 with Somatostatin.

Somatostatin (1.1 mg) obtained in Sigma Aldrich was dissolved in phosphate buffered saline (pH 7.2; 1 ml). The NHS functionalized polymer of Example 4 (Sample IV) (2 mg) was dissolved in a solution containing 100 mM sodium borate buffer (pH 9.3; 0.2 ml) and the somatostatin solution (0.1 ml) and stirred at room temperature for 72 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a

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flow rate of 1 ml / min. (Figure 13 (31: conjugate; 32: somatostatin)).

Example 20. Conjugation of the branched polymer functionalized with aldehyde of Example 8 with EPO.

The aldehyde functionalized polymer of Example 8 (2.5 mg) was dissolved in 80 mM sodium cyanoborohydride (1 ml) prepared in 100 mM sodium acetate buffer at pH 4.6. Erythropoietin (EPO), 0.2 ml, figure 1) obtained from a transgenic chicken (for example, as described in US Patent No. 6,730,822) in 100 mM sodium phosphate buffer (pH 6 , 98) at a concentration of 0.44 mg / ml was added to 0.3 ml of the polymer and stirred at room temperature for 72 hours. The resulting reaction mixture was analyzed on a Shodex KW-803 size exclusion column using a Beckman Coulter System Gold or Agilent 1100 series HPLC system at 280 nm with a flow rate of 1 ml / min. (Figure 14 (33: conjugate; 34: EPO; 35: buffer)).

Example 21. Preparation of poly-HEMA-PC linear conjugates through ATRP using 3- bromopropionic acid as initiator.

to. Polymerization reaction

The controlled polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water can be carried out using 3-bromopropionic acid (or a salt thereof, for example, a sodium salt) as a radical polymerization initiator by atom transfer (ATRP). The 3-bromopropionic acid initiator (153 mg, 1 mmol) is dissolved in sufficient distilled deionized water (about 15 to about 50 ml) in a sealed reaction flask equipped with a stirring apparatus. After purging the flask with nitrogen, the Cu (I) Br catalyst (1.4 mg, 1.0 mmol) and bipyridine (bpy) ligand (320 mg, 2.0 mmol) are added and the solution is stirred in an atmosphere of nitrogen. Then, the monomer, 2-methacryloxyethyl-2'- (trimethylammonium) ethyl phosphate, is added as a solid to the reaction mixture under a nitrogen atmosphere. The molecular weight of the desired product determines the amount of monomer used as indicated in the table below.

 Average molecular weight (Daltons)

 Amount of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate (calculated for internal salt)

 1,000

 0.852 g, 2.9 mmol

 2,000

 1,848 g, 6.3 mmol

 5,000

 4.84 g, 16.5 mmol

 10,000

 9.8 g, 33 mmol

 20,000

 19.8 g, 67.5 mmol

 40,000

 39.7 g, 135 mmol

The reaction mixture immediately becomes dark green and progressively more viscous. A temperature rise of approximately 2-4 ° C typically indicates that polymerization is occurring. After the reaction is complete, the resulting homopolymer bearing a free reactive carboxyl group can be precipitated by the addition of THF, redissolved in water, and chromatographed on a silica gel column to remove the residual ATRP catalyst. Alternatively, when it is desirable to couple the homopolymeric product, conjugation of the homopolymeric product can be performed without THF precipitation and chromatography as described below.

b. Conjugation reactions.

The conjugation of the homopolymeric product prepared as above in part 2a with a biologically active agent through the carboxylic acid of the propionic acid group derived from the 3-bromopropionic acid initiator can be carried out by using a suitable dehydrating agent, including, but not limitation, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), with or without the formation of an N-hydroxysuccinimide ester intermediate (NHS).

Homopolymeric products containing EDC-activated carboxyl or homopolymeric products containing NHS esters can be prepared by the addition of a buffering agent (eg, HEPES) to adjust the pH of an aqueous solution of the carboxyl-containing homopolymeric polymer product described. at 2a at approximately pH 7.2. EDC (from about 0.95 to about 0.98 mmol per 1 mmol of carboxyl-containing polymer) is introduced to form an EDC activated carboxylic acid. When it is considered desirable to form an NHS ester, 1.1 mmol of N-hydroxysuccinimide per mmol of the carboxyl-containing polymer is added after the addition of EDC. After formation, the NHS esters can be isolated for later use, either by freeze-drying the precipitation with a suitable solvent (for example, THF)

followed by lyophilization of the precipitate. Since NHS esters are generally more stable to hydrolysis at pH 5-6, where the NHS ester should be isolated, it is generally desirable to reduce the pH before precipitation and / or lyophilization of the NHS ester, which is performed in a desalination column equilibrated in a buffer of pH 4-pH 5.

5 Conjugates of biologically active agents carrying a primary amine (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alfa, beta interferon, human growth hormone and imiglucerase) with a polymeric product that carries a carboxylic acid activated by EDC or an NHS ester, they can be performed in a suitable buffer such as HEPES at pH 5-8 preferably at about pH

7.0. For the reaction, a small excess of the biologically active agent is introduced (for example, 1.1 mmol of the agent per mmol of the polymeric material). The reaction can be performed at room temperature, however, the reaction is typically preferred overnight (12 hours) on ice. When the biologically active agent has more than one reactive amine, it may be preferable to introduce the polymer carrying activated carboxylic acid into an excess of the biologically active agent to limit the formation of molecules having more than one polymer group associated with a single biologically active agent. active.

fifteen

Preparation of poly-HEMA-PC protein / polypeptide conjugates using 3-bromopropionic acid as an initiator.

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 1,000

 erythropoietin

 2,000

 erythropoietin

 5,000

 erythropoietin

 10,000

 erythropoietin

 20,000

 erythropoietin

 40,000

 erythropoietin

 1,000

 granulocyte colony stimulating factor

 2,000

 granulocyte colony stimulating factor

 5,000

 granulocyte colony stimulating factor

 10,000

 granulocyte colony stimulating factor

 20,000

 granulocyte colony stimulating factor

 40,000

 granulocyte colony stimulating factor

 1,000

 interferon alfa

 2,000

 interferon alfa

 5,000

 interferon alfa

 10,000

 interferon alfa

 20,000

 interferon alfa

 40,000

 interferon alfa

 1,000

 interferon beta interferon beta

 2,000

 beta interferon

 5,000

 beta interferon

 10,000

 beta interferon

 20,000

 beta interferon

 40,000

 beta interferon

 1,000

 human growth hormone

 2,000

 human growth hormone

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 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 5,000

 human growth hormone

 10,000

 human growth hormone

 20,000

 human growth hormone

 40,000

 human growth hormone

 1,000

 imiglucerase

 2,000

 imiglucerase

 5,000

 imiglucerase

 10,000

 imiglucerase

 20,000

 imiglucerase

 40,000

 imiglucerase

Conjugates of biologically active agents carrying a hydroxyl group or sulfhydryl groups can also be prepared. The reaction of a biologically active agent carrying a hydroxyl group or a sulfhydryl group with a polymeric product containing an EDC activated carboxylic acid will form a covalent conjugate having the biologically active agent attached to the polymeric group via an ester bond or a thioester bond. respectively.

The preparation of poly-HEMA-PC conjugates and biologically active agents in some cases can be performed in "single stage reactions". In this example, the ATRP reaction can be carried out in an aqueous solution followed by the addition of a suitable buffer (eg, HEPES) and a water-soluble dehydrating agent (EDC) to form a poly-HEMA-PC polymer carrying an activated carboxylic acid. The subsequent addition of a biologically active agent that carries a primary amine to the activated polymer forms a conjugate with the polymer through an amide bond. C. Purification and determination of molecular weight.

Conjugates prepared as in section 2b are subjected to Superdex 75 chromatography in normal saline (0.9 g of NaCl per 100 ml) or in a phosphate buffered saline solution (PBS), any of which are pharmaceutically acceptable excipients . The molecular weight is determined by the elution volume against the calibration standards available from the commercial suppliers included. Instead of chromatography, dialysis can be used to remove unwanted contaminants of low molecular weight.

Example 22. Preparation of poly-HEMA-PC conjugates through ATRP using an N-hydroxysuccinimide ether as initiator.

to. Preparation of N-hydroxysuccinimide ester initiators.

N-hydroxysuccinimide esters (NHS esters) can be purchased or prepared from the corresponding halogenated carboxylic acids by reacting an aqueous solution of the carboxylic acid with a dehydrating agent, such as EDC followed by the addition of N-hydroxysuccinimide. The N-hydroxysuccinimide bromoacetic acid ester initiator, which is commercially available from Sigma-Aldrich, can also be prepared by reacting an aqueous solution of bromoacetic acid with EDC followed by the addition of N-hydroxysuccinimide.

b. ATRP reactions using NHS esters.

NHS esters of carboxylic acids, for example, alkyl carboxylic acids, which carry one or more bromine, chlorine or iodine atoms can be used as initiators in ATRP reactions as long as they do not contain chemical groups that interfere with the polymerization reaction. By reaction, the polymerization of 2- methacryloyloxyethyl-2 '- (trimethylammonium) ethyl phosphate can be carried out using N-hydroxysuccinimide ester of bromoacetic acid as initiator of atom transfer radical polymerization (ATRP). The initiator (1 mmol) is dissolved in sufficient distilled deionized water that has been cooled on ice (approximately 15 ml) in a sealed reaction flask equipped with a stirring apparatus. After purging the flask with nitrogen, the Cu (I) Br catalyst (1.4 mg, 1.0 mmol) and bipyridine (bpy) ligand (320 mg, 2.0 mmol) are added and the solution is stirred in an atmosphere of nitrogen. Immediately before use. the monomer, 2-methacryloxyethyl-2'-trimethylammonioethyl phosphate is diluted in sufficient water and the pH is adjusted to approximately pH 4.5 with HCl or NaOH as necessary. The monomer solution is cooled in ice and added to the reaction mixture, which is stirred under a nitrogen atmosphere. The molecular weight of the desired product determines the amount of monomer used as indicated in the previous example.

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After the monomer is added, the reaction mixture becomes dark green and progressively more viscous. A temperature rise of approximately 2-4 ° C indicates that polymerization is occurring. While reactions are typically performed on ice, for some monomeric and initiator combinations it may be necessary to raise the temperature slightly or even heat the mixture to room temperature for up to 10 minutes. After the reaction is complete, the reaction mixture is cooled on ice.

C. Conjugation reactions.

After the polymerization reaction in part b, the product can be reacted with an amine containing a biologically active agent to form a conjugate having the polymeric portion of the conjugate bound to the biologically active agent via an amide bond. When the biologically active agent is stable at pH 4 at pH 5, it can be added to the reaction mixture before adjusting the pH in the range of about pH 7.0 to about pH 7.5 by adding a diluted base (by example, NaOH solution) or a suitable buffer, thus maintaining the NHS ester in the maximum stability range until the active agent is present for conjugation. Alternatively, the reaction mixture of 3b can be adjusted in the range of about pH 7.0 to about pH 7.5 and then the biologically active agent is immediately added to the reaction mixture. After incubation sufficient for the biologically active agent to be coupled to the polymeric agent, the reaction mixture is subjected to Superdex chromatography to separate the desired product from small molecule contaminants (e.g., non-polymerized monomers, catalyst and bipyridine) and the unreacted biologically active agent. When the biologically active agent is of sufficient mass, polymer compounds that are not coupled to the biologically active agent can also be separated from the conjugated biologically active agent. In addition, ion exchange chromatography can be used to separate conjugated biologically active agents, such as polymer conjugates from proteins and polypeptides, from unconjugated biologically active agents and polymeric compounds that are not coupled to a biologically active agent.

Preparation of poly-HEMA-PC protein / polypeptide conjugates using NHS esters of 3- bromoacetic acid as an initiator.

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 1,000

 erythropoietin

 2,000

 erythropoietin

 5,000

 erythropoietin

 10,000

 erythropoietin

 20,000

 erythropoietin

 40,000

 erythropoietin

 1,000

 granulocyte colony stimulating factor

 2,000

 granulocyte colony stimulating factor

 5,000

 granulocyte colony stimulating factor

 10,000

 granulocyte colony stimulating factor

 20,000

 granulocyte colony stimulating factor

 40,000

 granulocyte colony stimulating factor

 1,000

 interferon alfa

 2,000

 interferon alfa

 5,000

 interferon alfa

 10,000

 interferon alfa

 20,000

 interferon alfa

 40,000

 interferon alfa

 1,000

 interferon beta interferon beta

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35

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 2,000

 beta interferon

 5,000

 beta interferon

 10,000

 beta interferon

 20,000

 beta interferon

 40,000

 beta interferon

 1,000

 human growth hormone

 2,000

 human growth hormone

 5,000

 human growth hormone

 10,000

 human growth hormone

 20,000

 human growth hormone

 40,000

 human growth hormone

 1,000

 imiglucerase

 2,000

 imiglucerase

 5,000

 imiglucerase

 10,000

 imiglucerase

 20,000

 imiglucerase

 40,000

 imiglucerase

Example 23. Preparation of linear poly-HEMA-PC conjugates through ATRP using 3- bromopropionaldehyde dimethylacetal as initiator.

to. Polymerization reaction and aldehyde deprotection.

Polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water is carried out using an ATRP reaction as set forth in Example 21, part 2a by replacing 3-bromopropionaldehyde dimethylacetal as initiator instead of 3-bromopropionic acid. As with the products in example 2, the monomer ratio with respect to the initiator used will determine the molecular weight of the product, and products with molecular weights of 1,000, 2,000, 5,000, 10,000, 20,000, and 40,000 are prepared using ratios of suitable monomer-initiator. Once the polymerization reaction is complete, dimethylacetyl can be converted to an aldehyde by exposure to an aqueous acid (for example, aqueous HCl or acetic acid). When necessary, unused monomers and other polymerization reaction reagents can be removed from polymeric products carrying aldehyde on a desalting gel column or by chromatography, for example, in Superdex. When the aldehyde is to be coupled to an amine, the chromatographic media can be equilibrated with a suitable buffer such as borate buffer at pH 9.5.

b. Conjugation reactions.

Products carrying aldehyde 4a can be reacted with a primary amine containing biologically active agents to form an imine bond. Primary amines that contain biologically active agents include polypeptides and proteins that contain an accessible amino group in a naturally occurring amino acid, or those in which a primary amine has been introduced in order to couple them to the polymeric agent. The imine bond formation is performed in borate buffer at pH 9.5 or in other suitable imine bond formation conditions. Proteins or polypeptides, including, but not limited to, erythropoietin, granulocyte colony stimulating factor, alpha interferon, beta interferon, human growth hormone, and imiglucerase, can be reacted with polymers containing aldehydes of any molecular weight set forth in 4a for form conjugates that have hydrolytically susceptible bonds. When desirable, the imine can be reduced in an amine to avoid hydrolysis of that conjugate bond.

Example 24. Preparation of poly-HEMA-PC linear conjugates using maleimide conjugation of biologically active agents containing sulfhydryl.

to. Polymerization reactions

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65

Polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water is carried out using an ATRP reaction as set forth in Example 21, part 2a, substituting 3-bromopropanamine as initiator instead of 3-bromopropionic acid. As with the products in Example 21, the ratio of monomer to initiator employed will determine the molecular weight of the product, and products with molecular weights of 1,000,

2,000, 5,000, 10,000, 20,000, or 40,000, are prepared using suitable monomer-initiator ratios. After completion of the polymerization reaction, the polymerization reaction reagents (for example, catalyst, ligand, unpolymerized monomers) can be removed from the polymer products containing aldehyde in a desalting gel column or by chromatography, for example, in Superdex When the polymer product containing amines is to be coupled to maleimidyl functionality through the use of a bifunctional agent containing an NHS ester, the chromatographic media can be equilibrated with a suitable buffer such as phosphate, bicarbonate / carbonate HEPES or buffers. borate having a pH of approximately pH 7 to approximately pH 9.

b. Introduction of maleimide functionality through the use of a bifunctional agent.

Polymeric amine-containing products of any molecular weight prepared in 5a are suspended in 50 mM phosphate buffer at pH 7.5 and cooled to 4 ° C. A 1,2 molar excess of 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfo-succinimidyl (Sulfo-SMCC, available in Pierce) is introduced and the reaction is allowed to continue for 8 hours at 4 ° C. Alternatively, the reaction can be started at 4 ° C and stirred for 1-2 hours at that temperature and then gradually heated to room temperature. The reaction may be followed by the release of N-hydroxysuccinimide at 260 nm.

Once prepared, polymers bearing maleimide can be reacted immediately with biologically active agents containing sulfhydryl. Optionally, the products can be prepared for later use by precipitation with an organic solvent such as tetrahydrofuran (THF) and lyophilization or subjected to purification by dialysis or chromatography on a desalination gel (eg, Biogel P6DG) and lyophilization.

C. Conjugation with biologically active agents that carry sulfhydryl.

Products bearing maleimide of any molecular weight from 5b can be reacted with biologically active agents containing sulfhydryl to form an imine bond. Biologically active agents containing sulfhydryl, including polypeptides and proteins containing cysteine residues, and those in which cysteines have been introduced in order to form conjugates, including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alpha, beta interferon, human growth hormone, and imiglucerase. When necessary or desirable, sulfhydryl groups can be introduced during the preparation of proteins or polypeptides by chemical or biochemical means that can introduce cysteine or other amino acids containing sulfhydryl, or alternatively, by subsequent chemical modification with agents that introduce sulfhydryl groups (by example, 2-iminothiolane).

Biologically active agents carrying a sulfhydryl group can be introduced into the reaction mixture prepared in 5b after the reaction of the Sulfo-SMCC bifunctional agent with the polymer carrying the amine prepared in 5a. Alternatively, when a biologically active agent carrying sulfhydryl contains neither interfering primary amines nor other groups that can react with the Sulfo-SMCC NHS ester, the biologically active agent can be introduced into the reaction mixture before, or simultaneously with, Sulfo-SMCC as selective coupling to both the NHS ester for an amine and maleimide for a sulfhydryl will occur under similar conditions (for example, a pH of about 7.0 to about 7.5).

d. Purification and determination of molecular weight.

As described in Example 21, part 2c, the conjugates can be purified and their molecular weight can be determined by chromatographic means. Chromatography of the conjugates prepared in 5b is performed in Superdex 75 or Superdex Peptide in normal saline or phosphate buffered saline (PBS), and the molecular weight is determined against chromatography calibration standards.

Example 25. Preparation of branched poly-HEMA-PC conjugates through ATRP using 2,3-dibromopropionic acid as an initiator.

to. Polymerization reactions

Polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water is carried out using an ATRP reaction as set forth in Example 21, part 2a, substituting 2,3-dibromopropionaldehyde dimethylacetal (1 mmol, 271 mg) as an initiator instead of 3-bromopropionic acid to form a branched 2-arm polymeric compound. As analyzed in Example 21, the molecular weight of the desired product determines the amount of monomer used as indicated in the table below.

 Average molecular weight (Daltons)

 Amount of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate (calculated for internal salt)

 5,000

 4.7 g, 16.1 mmol

 10,000

 9.7 g, 33.1 mmol

 20,000

 19.7 g, 67.1 mmol

 40,000

 39.7 g, 135 mmol

b. Conjugation reactions.

The conjugation of the branched homopolymeric product prepared as in part a with a biologically active agent through the carboxylic acid of the propionic acid group derived from the 2,3-dibromopropionic acid initiator can be performed by using a dehydrating / activating agent of suitable carboxylic acid (eg EDC) or through the formation of an intermediate N-hydroxysuccinimide (NHS) ester as described in Example 21, part b. After formation, the NHS esters can be reacted immediately with a biologically active agent carrying a primary amine or isolated for later use by lyophilization or precipitation with a suitable solvent (eg, THF) followed by lyophilization of the precipitate as is analyzed in Example 21, part b.

Conjugates of biologically active agents that carry a primary amine (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alpha, beta interferon, human growth hormone and imiglucerase) with branched polymer products that carry an acid EDC activated carboxylic acid or an NHS ester are prepared in a suitable buffer such as HEPES at pH 5-8 preferably at about pH 7.0 as described in Example 21, part b.

20 Preparation of poly-HEMA-PC protein / polypeptide conjugates using 2,3-dibromopropionaldehyde dimethylacetal as initiator.

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 5,000

 erythropoietin

 10,000

 erythropoietin

 20,000

 erythropoietin

 40,000

 erythropoietin

 5,000

 granulocyte colony stimulating factor

 10,000

 granulocyte colony stimulating factor

 20,000

 granulocyte colony stimulating factor

 40,000

 granulocyte colony stimulating factor

 5,000

 interferon alfa

 10,000

 interferon alfa

 20,000

 interferon alfa

 40,000

 interferon alfa

 5,000

 beta interferon

 10,000

 beta interferon

 20,000

 beta interferon

 40,000

 beta interferon

 5,000

 human growth hormone

 10,000

 human growth hormone

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fifty

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 20,000

 human growth hormone

 40,000

 human growth hormone

 5,000

 imiglucerase

 10,000

 imiglucerase

 20,000

 imiglucerase

 40,000

 imiglucerase

As described in Example 21, part b, conjugates of biologically active agents bearing a hydroxyl group or a sulfhydryl group can also be prepared. The reaction of a biologically active agent carrying a hydroxyl group or a sulfhydryl group with a branched polymeric product carrying an activated carboxylic acid (e.g., an activated carboxy with EDC) will form a covalent conjugate having the biologically active agent attached to the group. polymeric via an ester bond or a thioester bond, respectively.

The preparation of branched poly-HEMA-PC and biologically active conjugates by "one-step reactions" is also possible with branched chain polymers. C. Purification and determination of molecular weight.

Purification and molecular weight determination are performed as described in Example 21, part c.

Example 26. Preparation of branched poly-HEMA-PC conjugates through ATRP using 3- bromopropionaldehyde dimethylacetal as initiator.

to. Polymerization reaction and aldehyde deprotection.

Polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water is carried out using an ATRP reaction as set forth in Example 21, part to replacing 2,3-dichloropropionaldehyde or its dimethylacetal (1 mmol) as an initiator instead of 3-bromopropionic acid to form a branched 2-arm polymeric compound. As with the branched polymer products in example 25, the ratio of monomer to the initiator used will determine the molecular weight of the product, and products with molecular weights of 5,000, 10,000,

20,000, and 40,000, are prepared using suitable monomer-initiator ratios. After completion of the polymerization reaction, if dimethyl acetal has been used, the dimethylacetyl group is converted to an aldehyde by exposure to aqueous acid (for example, aqueous HCl or acetic acid). Unused monomers and other polymerization reaction reagents that include the catalyst can be removed from polymer products containing aldehyde by dialysis, or by chromatography or Superdex desalination gel column. When the aldehyde is to be coupled to an amine, the chromatographic media can be equilibrated with a suitable buffer such as borate buffer at pH 9.5.

b. Conjugation reactions.

Conjugates of biologically active agents carrying a primary amine (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alpha, beta interferon, human growth hormone and imiglucerase) are formed with branched polymer products of any weight molecular prepared in part a. The branched polymer products carrying aldehyde of the part a are reacted with the biologically active agents containing primary amine to form an imine bond as described in Example 23, part a. When desirable, the imine can be reduced in an amine to prevent hydrolysis of that conjugate bond as described in Example 23, part b.

C. Purification and determination of molecular weight.

Molecular weight and purification determinations, particularly when the imine bond has been reduced in an amine with a borohydride reagent, are performed as described in Example 21, part c.

Example 27. Preparation of poly-HEMA-PC branched conjugates using maleimide conjugation of biologically active agents containing sulfhydryl.

Polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water is carried out using an ATRP reaction as set forth in Example 24, part a, substituting 1,3-dibromo-2-propanamine as a starter instead. of 3-bromopropionic acid to prepare a branched polymer with two arms. As with the products in examples 21 and 24, the ratio of monomer to initiator employed will determine the molecular weight of the

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Four. Five

product, and products with molecular weights of 5,000, 10,000, 20,000, or 40,000, are prepared using suitable monomer-initiator ratios. Maleimide functionalities can be introduced into the branched chain polymeric compound to form polymeric compounds containing branched chain maleimide through the use of bifunctional agents (e.g., Sulfo-SMCC) as described in Part b of Example 24. Polymeric compounds containing branched chain maleimide can be prepared for later as described in part 5b. Alternatively, once prepared, polymeric compounds containing branched chain maleimide can be reacted with biologically active agents containing sulfhydryl to form conjugates. Conjugates of biologically active agents carrying a sulfhydryl (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alfa, beta interferon, human growth hormone and imiglucerase) with polymeric compounds containing branched chain maleimide of 5,000 , 10,000, 20,000 or 40,000 Daltons are prepared as described in Example 24, parts c and d.

Example 28. Preparation of bifurcated poly-HEMA-PC conjugates through ATRP using DL-bromosuccinic acid as initiator.

to. Polymerization reactions

Polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water is carried out using an ATRP reaction as set forth in Example 21, part a, substituting DL-bromosuccinic acid (bromobutanedioic acid, Acros Organics) as an initiator instead of 3-bromopropionic acid. The reaction forms a dicarboxylic polymer product that carries the carboxyl groups in the succinic acid group derived from the initiator. The molecular weight of the desired product determines the amount of monomer employed, and polymeric molecules having an average molecular weight of 1,000, 2,000, 5,000, 10,000, 20,000, and 40,000 Daltons are prepared using suitable monomer-initiator ratios.

b. Conjugation reactions.

The conjugation of the dicarboxylic polymer product prepared as in part a with a biologically active agent through the carboxylic acid groups is carried out by the use of a dehydration agent / carboxylic acid activator (eg, EDC) or through the formation of an intermediate N-hydroxysuccinimide (NHS) ester as described in Example 21 part b, adjusting the amount of reagents to account for the 2 moles of carboxyl functionalities present per mole of dicarboxylic polymer product. When NHS esters are prepared, they can be reacted immediately with a biologically active agent carrying a primary amine or can be isolated for later use by lyophilization or precipitation with a suitable solvent (eg, THF) followed by lyophilization of the precipitate as is analyzed in Example 21, part b.

Conjugates of biologically active agents that carry a primary amine (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alpha, beta interferon, human growth hormone and imiglucerase) with the dicarboxylic polymer product that carries a carboxylic acid EDC-activated or NHS ester groups are prepared in a suitable buffer such as HEPES at pH 5-8 preferably at about pH 7.0 as described in Example 21, part b.

Preparation of poly-HEMA-PC bifurcated protein / polypeptide conjugates using DL-bromosuccinic acid as an initiator.

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 5,000

 erythropoietin

 10,000

 erythropoietin

 20,000

 erythropoietin

 40,000

 erythropoietin

 5,000

 granulocyte colony stimulating factor

 10,000

 granulocyte colony stimulating factor

 20,000

 granulocyte colony stimulating factor

 40,000

 granulocyte colony stimulating factor

 5,000

 interferon alfa

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 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 10,000

 interferon alfa

 20,000

 interferon alfa

 40,000

 interferon alfa

 5,000

 beta interferon

 10,000

 beta interferon

 20,000

 beta interferon

 40,000

 beta interferon

 5,000

 human growth hormone

 10,000

 human growth hormone

 20,000

 human growth hormone

 40,000

 human growth hormone

 5,000

 imiglucerase

 10,000

 imiglucerase

 20,000

 imiglucerase

 40,000

 imiglucerase

As described in part 2b, conjugates of biologically active agents carrying a hydroxyl group or a sulfhydryl group can also be prepared. The reaction of a biologically active agent carrying a hydroxyl group or a sulfhydryl group with a branched polymer product bearing an activated carboxylic acid group (e.g., EDC activated carboxyls) will form bifurcated covalent conjugates having the biologically active agents bound to the group. polymeric by ester bonds or thioester bonds, respectively.

The preparation of bifurcated poly-HEMA-PC conjugates and biologically active agents by "one-step reactions" is also possible with the dicarboxylic polymer products prepared as in part a. C. Purification and determination of molecular weight.

Purification and molecular weight determination are performed as described in Example 21, part c.

Example 29. Preparation of branched poly-HEMA-PC conjugates through ATRP using 2- chloroglutaraldehyde as initiator.

to. Polymerization reaction and aldehyde deprotection.

Polymerization of 2-methacryloxyethyl-2 '- (trimethylammonium) ethyl phosphate in water is carried out using an ATRP reaction as set forth in Example 21, part 2a replacing 2-chloroglutaraldehyde (see U.S. Patent 6,703,528) or its dimethylacetal as an initiator instead of 3-bromopropionic acid. As with the polymer products in Example 21, the monomer-initiator ratio employed will determine the molecular weight of the polymer product carrying dialdehyde. Polymeric dialdehyde products with molecular weights of 1,000,

2,000, 5,000, 10,000, 20,000, and 40,000 are prepared using suitable monomer-initiator ratios. After completion of the polymerization reaction, if dimethyl acetal has been used, the dimethylacetyl group is converted to an aldehyde by exposure to aqueous acid (for example, aqueous HCl or acetic acid). Unused monomers and other polymerization reaction reagents that include the catalyst can be removed from the dialdehyde polymer product by dialysis, or by chromatography or Superdex desalination gel column. When a polymeric dialdehyde product is to be coupled to an amine, the chromatographic media can be equilibrated with a suitable buffer such as borate buffer at pH 9.5.

b. Conjugation reactions.

Conjugates of biologically active agents that carry a primary amine (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alpha, beta interferon, human growth hormone and imiglucerase) are formed with the dialdehyde polymer products of any molecular weight prepared in part a. The polymeric dialdehyde products of part a are reacted with the biologically active agents containing primary amine to form imine bonds as described in Example 23, part by adjusting the two moles of aldehyde per mole of polymeric dialdehyde product. When desirable,

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30

35

40

Four. Five

fifty

55

60

The mine bonds can be reduced in an amine to avoid hydrolysis of that conjugate bond as described in Example 23, part b.

C. Purification and determination of molecular weight.

Molecular weight and purification determinations, particularly when the imine bond has been reduced in amines with a borohydride reagent, are performed as described in Example 21, part c.

Example 30. Preparation of bifurcated poly-HEMA-PC conjugates using maleimide conjugation of biologically active agents containing sulfhydryl.

The polymerization of phosphate of 2-methacryloxyletl-2 '- (trimetalamine) or water is carried out using an ATRP reaction as set forth in Example 21, part a , substituting 1,3-diamino-2-bromopropane as an initiator instead of 3-bromopropionic acid to prepare a bifurcated compound. As with the products in examples 21 and 24, the monomer ratio to the initiator employed will determine the molecular weight of the product, and products with molecular weights of 5,000, 10,000, 20,000, or 40,000, are prepared using monomer ratios. suitable initiator. Maleimide functionalities can be introduced into the amine groups through the use of a bifunctional agent (for example, Sulfo-SMCC) as described in Example 24 Part b. Bifurcated polymeric compounds having two maleimide reactive groups can be prepared for later as described in Example 24, part b. Alternatively, once prepared, polymeric compounds containing maleimide can be reacted with biologically active agents containing sulfhydryl to form conjugates. Conjugates of biologically active agents carrying a sulfhydryl (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alfa, beta interferon, human growth hormone and imiglucerase) with polymeric compounds containing branched chain maleimide of 5,000 , 10,000, 20,000 or 40,000 Daltons are prepared as described in part 5c and 5d. The conjugates of FAb2 antibody fragments, which can be reduced with mercaptoethanol or DTT (dithiothreitol) to produce a single sulfhydryl group at the end of each of the resulting FAb fragments, are prepared in the same manner. The bifurcated conjugates of the FAb fragments are particularly useful as "pseudo-antibody constructs".

Example 31. Preparation of branched and branched poly-HEMA-PC conjugates through ATRP using 2,3-dibromosuccinic acid as an initiator.

to. Polymerization reactions

Polymerization of phosphate of 2-methacryloxyletl-2 '- (trimetalamine) or water is carried out using an ATRP reaction as set forth in Example 21, part 2a substituting 2,3-dibromosuccinic acid as an initiator instead of 3-bromopropionic acid. The reaction forms a dicarboxylic acid product having two polymer arms that carry the carboxyl groups in the succinic acid group derived from the initiator. The molecular weight of the desired product determines the amount of monomer used, and polymeric molecules having an average molecular weight of 5,000, 10,000, 20,000 and 40,000 dalton are prepared using suitable monomer-initiator ratios.

b. Conjugation reactions.

The conjugation of the dicarboxylic acid products having two polymer arms prepared as in part 12a with a biologically active agent through the carboxylic acid groups is carried out by the use of a dehydrating / activating agent of carboxylic acid (for example , EDC) or by forming an intermediate N-hydroxysuccinimide ester (NHS) as described in Example 21 part 2b by adjusting the amount of reagents to account for the 2 moles of carboxyl functionalities present per mole of dicarboxylic polymer product. When NHS esters are prepared, they can be reacted immediately with a biologically active agent carrying a primary amine or can be isolated for later use by lyophilization or precipitation with a suitable solvent (eg, THF) followed by lyophilization of the precipitate as It is analyzed in part 2b.

The conjugates of biologically active agents that carry a primary amine (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, interferon alpha, beta interferon, human growth hormone and imiglucerase) with the dicarboxylic acid product that has two arms of polymer carrying an EDC activated carboxylic acid or NHS ester groups are prepared in a suitable buffer such as HEPES at pH 5-8 preferably at about pH 7.0 as described in Example 21, part b.

Preparation of branched and branched poly-HEMA-PC protein / polypeptide conjugates using 2,3-dibromosuccinic acid as an initiator.

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 5,000

 erythropoietin

 10,000

 erythropoietin

 20,000

 erythropoietin

 40,000

 erythropoietin

 5,000

 granulocyte colony stimulating factor

 10,000

 granulocyte colony stimulating factor

 20,000

 granulocyte colony stimulating factor

 40,000

 granulocyte colony stimulating factor

 5,000

 interferon alfa

 10,000

 interferon alfa

 20,000

 interferon alfa

 40,000

 interferon alfa

 5,000

 beta interferon

 10,000

 beta interferon

 20,000

 beta interferon

 40,000

 beta interferon

 5,000

 human growth hormone

 10,000

 human growth hormone

 20,000

 human growth hormone

 40,000

 human growth hormone

 5,000

 imiglucerase

 10,000

 imiglucerase

 20,000

 imiglucerase

 40,000

 imiglucerase

As described in part 2b, conjugates of biologically active agents carrying a hydroxyl group or a sulfhydryl group can also be prepared. The reaction of a biologically active agent carrying a hydroxyl group or a sulfhydryl group with a dicarboxylic acid product having two polymer arms bearing an activated carboxylic acid group (e.g., EDC activated carboxyls) will form bifurcated covalent conjugates that they have the biologically active agents bound to the two-arm polymer group by ester bonds or thioester bonds, respectively.

The preparation of branched and branched poly-HEMA-PC conjugates and biologically active agents by "one-step reactions" is also possible with the dicarboxylic acid product having two polymeric arms prepared as in part a. C. Purification and determination of molecular weight.

Purification and molecular weight determination are performed as described in Example 21, part c.

fifteen

Example 32. Preparation of branched and branched poly-HEMA-PC conjugates through ATRP using 3,3-dichloroglutaric acid as an initiator.

The branched and bifurcated polymer conjugates of poly-HEMA-PC through ATRP can be prepared as in Example 31 using 3,3-dichloroglutaric acid as the initiator instead of dibromosuccinic acid. Conjugates of biologically active agents that carry a primary amine (including, but not limited to, erythropoietin, granulocyte colony stimulating factor, alpha interferon, beta interferon, growth hormone

human and imiglucerase) with the dicarboxylic acid product having two polymer arms of any molecular weight described in Example 31 can be formed.

Preparation of branched and bifurcated poly-HEMA-PC protein / polypeptide conjugates using 3.35 dichloroglutaric acid as an initiator.

 Average molecular weight of the polymer portion of the conjugate (Daltons)

 Biologically Conjugated Active Agent

 5,000

 erythropoietin

 10,000

 erythropoietin

 20,000

 erythropoietin

 40,000

 erythropoietin

 5,000

 granulocyte colony stimulating factor

 10,000

 granulocyte colony stimulating factor

 20,000

 granulocyte colony stimulating factor

 40,000

 granulocyte colony stimulating factor

 5,000

 interferon alfa

 10,000

 interferon alfa

 20,000

 interferon alfa

 40,000

 interferon alfa

 5,000

 beta interferon

 10,000

 beta interferon

 20,000

 beta interferon

 40,000

 beta interferon

 5,000

 human growth hormone

 10,000

 human growth hormone

 20,000

 human growth hormone

 40,000

 human growth hormone

 5,000

 imiglucerase

 10,000

 imiglucerase

 20,000

 imiglucerase

 40,000

 imiglucerase

Example 33. Taxol conjugates.

Taxol, which carries two hydroxyl groups, is conjugated to homopolymers bearing a free reactive carboxyl group prepared in Example 21, part 2a of 1,000, 2,000, 5,000, 10,000, 20,000 or 40,000 Daltons.

Taxol is conjugated to homopolymers dissolved in HEPES buffer at about pH 7.2 by adding from about 0.95 to about 0.98 mmol of EDC for every 1 mmol of homopolymer carrying a carboxyl group to form a carboxyl group activated by EDC in the homopolymer. To avoid having more than one taxol hydroxyl group conjugated to the homopolymer, the activated homopolymer is slowly added with stirring to a 1.2 molar excess of the taxol suspended in HEPES buffer. When a large amount of the polymer conjugate is to be prepared, small portions of homopolymer having an EDC activated carboxyl group can be prepared and reacted with taxol in sequence so that the EDC activated carboxyl group 20 (an intermediate of o-acylisourea) does not decompose before the sum. Temperature reduction also reduces the rate of hydrolysis of the carboxyl group activated by EDC. As conjugation progresses, the suspended taxol gradually dissolves.

Claims (32)

5 10 fifteen twenty 25 30 35 1. A compound comprising a biologically active agent covalently bonded to a branched polymer containing phosphorylcholine, wherein the polymer has two or more polymer arms extending from a single group, wherein the polymeric portion of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl phosphate]. 2. The compound of claim 1, wherein the phosphorylcholine-containing polymer is covalently linked to a biologically active agent through a binding group and an optional spacer group. 3. The compound of claim 1, wherein the phosphorylcholine-containing polymer is covalently linked to at least one of an amino group, a hydroxyl group, a sulfhydryl group and a carboxyl group of the biologically active agent. 4. The compound of claim 1, wherein the branched polymer has 2 polymer arms, 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 8 polymer spleens or more than 8 spleens of polymer. 5. The compound of claim 4, wherein the polymer has 3 polymer spleens or 4 polymer spleens. 6. The compound of claim 1 having the formula (IVb), image 1 where the polymeric portions: image2 of the compound are poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate; each occurrence of E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), -N (^ -4 alkyl) 2, -OH, -O- (C1- alkyl 4), and -O- (C1-4 alkyl substituted with fluorine); Sp1 is selected from the group consisting of: -C12-12 alkyl-, -C3-12 alkyl-, - (C1-8 alkyl) - (C3-12 cycloalkyl) - (C0-8 alkyl) -, - (CH2) 1-12O-, (- (CH2) -O- (CH2) 1-6-) 1-12-, (- (CH2) 1-4-NH- (CH2) 1-4) 1-12-, ( - (CH2) 1-4-O- (CH2) 1-4) 1-12-, (- (CH2) 1-4-O- (CH2) 1-4-) 1-12O- (CH2) 1- 12-, - (CH2) 1-12- (C = O) -O-, - (CH2> m2-O- (C = O) -, - (phenyl) - (CH2) 1-3- (C = O) -O-, - (phenyl) - (CH2) 1-3- (C = O) -NH-, - (C1-6 alkyl) - (C = O) -O- (C0-6 alkyl) - , - (CH2> m2- (C = O) -O- (CH2) 1-12-, -CH (OH) -CH (OH) - (C = O) -O- - CH (OH) -CH ( OH) - (C = O) -NH-, -S-maleimido- (CH2) 1-6-, -S-maleimido- (C1-3 alkyl) - (C = O) -NH-, -S-maleimido - (C1-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) -, - (C1-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -O-, - (C1-3 alkyl) - (cycloalkyl 5 10 fifteen twenty 25 30 35 40 C5-6) - (C0-3 alkyl) - (C = O) -NH-, -S-maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) -, - (C0-3 alkyl) -phenyl- (C = O) -NH-, - (CH2) i- i2-NH- (C = O) -, - (CH2) i- i2- (C = O) -NH-, - (phenyl) - (CH2) i -3- (C = O) -NH-, -S- (CH2) - (C = O) -NH- (phenyl) -, - (CH2) i-i2- (C = O) - NH- (CH2) i-i2-, - (CH2) 2- (C = O) -O- (CH2) 2-O- (C = O) - (CH2) 2- (C = O) -NH -, - (Ci-a alkyl) - (C = O) -N- (Ci-a alkyl) -, acetal, ketal, acyloxyalkyl ether, -N = CH-, - (Ci-6 alkyl) -SS- ( C0-6 alkyl) -, - (Ci-6 alkyl) -SS- (Ci-6 alkyl) - (c = O) -O-, - (Ci-a alkyl) -SS- (Ci-6 alkyl) - (C = O) -NH-, -SS- (CH2) i-3- (C = O) -NH- (CH2) i-4-NH- (C = O) - (CH2) i-3-, -SS- (CcwMphenyl alkyl) -, -SS- (Ci-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) i-5-, - (Ci-3 alkyl) - (phenyl) - (C = O) -NH- (CH2) i-5- (C = O) -NH-, -SS- (Ci-3 alkyl) -, - (Ci-3 alkyl) - (phenyl) - (C = O) -NH-, -O- (Ci-Ca alkyl) -S (O2) - (Ci-a alkyl) -O- (C = O) -NH-, -SS- (CH2) i-3- (C = O) -, - (CH2) i- 3- (C = O) -NH-N = CSS- (CH2) i-3- (C = O) -NH- (CH2) i-5-, - (CH2) i-3- (C = O) -NH- (CH2) i-5- (C = O) -NH-, - (CH2) 0-3- (heteroaryl) - (CH2) 0-3-, - (CH2) 0-3-phenyl- (CH2) 0-3-, , R2 R2 - N “C— - (C] alkyl .fi) -C — N- (Ci.g alkyl) - R2 R2 \ ‘_ - (C | alkyl) - (arl) -C = N (C ^ g alkyl) - - (Cj.6 alkyl) -C = N- (aryl (- (C! _ * alkyl)) - OR Cj.g alkyl) —O — P — O— (alkyl Co.g) - OH; Y n is 0 or i, where when n is 0, Spi is a link; m is an integer that varies from 2 to 2,000; L is selected from the group consisting of Ci-4 alkyl, cycloalkyl, carboxy-Ci-4 alkyl, carboxycycloalkyl, Ci-4-alkoxyCi-4 alkyl and cyclic alkyl ether; A is a biologically active agent; Z is selected from the group consisting of: phosphate, phosphate ester, - (C = O) O-, carboxylic acid ester, thioester, amide, disulfide, amine, -NH (Ri) -, amidine, hydrazone, -N = CH-, image3 , -NH-C (Ri) H-, -S-CH2-C (OH) (R2) -, -O-CH2-C (OH) (R2) -, -C (= O) O-CH2-C (OH) (R2) -, - NR2-CH2-C (OH) (R2) -, -S-CH2- C (SH) (R2) -, -O-CH2-C (SH) (R2) -, -C (= O) O-CH2-C (SH) (R2) -, -NR2-CH2-C (SH) (R2) -, image4 -C (R2) H-NH-, - (C = O) -NH- (C = O) -NH-, R2 -C = N- (C = 0) -NH-, -C (R2) H-NH- (C = O) -NH-, - (C = O) -NH- (C = O) -O-, R; -C = N- (C = 0) -0- , -C (R2) H-NH- (C = O) -O-, - (C = O) -NH- (C = S) -NH-, R2 -C = N- (OS) -NH , -C (R2) H-NH- (C = S) -NH-, - (C = O) -NH- (C = S) -O-, 5 10 fifteen twenty 25 30 -C (R2) H-NH- (C = S) -O-, R; -C = N- (C = S) -0 R2 -C = N-NH , -NH- (C = O) -NH-, -O- (C = O) -NH-, -NH- (C = S) -NH-, -O- (C = S) -NH-, - S-CH2CH2- (C = O) -, -S-CH2CH (CH3) - (C = O) - S- CH2CH2- (C = O) O-, -S-CH2CH (CH3) - (C = O ) O-, -S-CH2CH2- (C = O) NH-, -S-CH2-CH2- (pyridyl) -, -S-CH2-CH2-SO2-, -S- CH2-CH2-SO2-, - S-CH2- (C = O) -O-, -S-CH2- (C = O) -NH-, -S-CH2- (C = O) -, and image5 R1 is C1.6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms; and R2 is H, C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms. 7. The compound of claim 1 having the formula (Vb), image6 where the polymeric portions: image7 of the compound are poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate; each occurrence of E is independently selected from the group consisting of Br, Cl, I, -NH2, -NH (C1-4 alkyl), -N (C1-4 alkyl) 2, -OH, -O- (C1 alkyl. 4), and -O- (C1.4 alkyl substituted with fluorine); Sp1 is selected from the group consisting of: -C12-12 alkyl-, -C3-12 alkylcyclo-, - (C1-C alkyl) - (C3-12 cycloalkyl) - (C0-8 alkyl) -, - (CH2) 1-12O-, (- (CH2) 1-6-O- (CH2) 1-6-) 1-12-, (- (CH2) 1-4-NH- (CH2) 1-4) 1-12 -, (- (CH2) -O- (CH2) 1-4) 1-12-O-, (- 5 10 fifteen twenty 25 30 35 40 (CH2) i-4-O- (CH2) i-4-) i-i2O- (CH2) i-i2-, - (CH2) i-i2- (C = O) -O-, - (CH2) i-i2-O- (C = O) -, - (phenyl) - (CH2) i-3- (C = O) -O-, - (phenyl) - (CH2) i-3- (C = O ) -NH-, - (Ci-6 alkyl) - (C = O) -O- (C0-6 alkyl) -, - (CH2) i-i2- (C = O) -O- (CH2) i- i2-, -CH (OH) - CH (OH) - (C = O) -O - CH (OH) -CH (OH) - (C = O) -NH-, -S-maleimido- (CH2) i-6-, -S-maleimido- (Ci-3 alkyl) - (C = O) -NH-, -S-maleimido- (Ci-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl ) -, - (Ci-3 alkyl) - (C5-6 cycloalkyl) - (C0-3 alkyl) - (C = O) -O-, - (Ci-3 alkyl) - (C5-6 cycloalkyl) - ( C0-3 alkyl) - (C = O) -NH-, -S-maleimido- (C0-3 alkyl) -phenyl- (C0-3 alkyl) -, - (C0-3 alkyl) - phenyl- (C = O) -NH-, - (CH2) i-i2-NH- (C = O) -, - (CH2) i-i2- (C = O) -NH-, - (phenyl) - (CH2) i - 3- (C = O) -NH-, -S- (CH2) - (C = O) - NH- (phenyl) -, - (CH2) i-i2- (C = O) -NH- (CH2) i-i2-, - (CH2) 2- (C = O) -O- (CH2) 2-O- (C = O) - (CH2) 2- (C = O) -NH-, - (Ci alkyl - 6) - (C = O) -N- (C1-6 alkyl) -, acetal, ketal, acyloxyalkyl ether, -N = CH-, - (Ci-6 alkyl) -SS- (C0-6 alkyl) - , - (C1-6 alkyl) - SS- (Ci-6 alkyl) - (C = O) -O-, - (Ci-6 alkyl) -SS- (Ci-a alkyl) - (C = O) - NH-, -SS- (CH2) i-3- (C = O) -NH - (CH2) i-4-NH- (C = O) - (CH2) i-3-, -SS- (C0-3 alkyl) - (phenyl) -, -SS- (Ci-3 alkyl) - ( phenyl) - (C = O) -NH- (CH2) i-5-, - (Ci.s alkyl) - (phenyl) - (C = O) -NH- (CH2) i-5- (C = O ) -NH-, -SS- (C1.3 alkyl) -, - (Ci-3 alkyl) - (phenyl) - (C = O) -NH-, -O- (Ci-C6 alkyl) -S (O2 ) - (Ci-6 alkyl) -O- (C = O) -NH-, -SS- (CH2) i-3- (C = O) -, - (CH2) i-3- (C = O) -NH-N = CSS- (CH2) i-3- (C = O) -NH- (CH2) i-5-, - (CH2) i-3- (C = O) -NH- (CH2) i -5- (C = O) -NH-, - (CH2) 0-3- (heteroaryl) - (CH2) 0-3-, - (CH2) 0-3-phenyl- (CH2) 0-3-, , R2 R2 - N ^ C— - (al9ui1 ^ 1 -6) -C — N- (alkyl C, .6) - R2 R2 \ »„ - (C- | .fi alkyl) - (arl) -C = N (C ^ g alkyl) - - (Cj.6 alkyl) -C = N- (aryl) - (C! _ * alkyl) - OR alkyl C] _é) ~ 0 — P — O— (alkyl Co_g) OH Y n is 0 or 1, where when n is 0, Sp1 is a link; m is an integer that varies from 2 to 2,000; L is selected from the group consisting of Ci-4 alkyl, cycloalkyl, carboxy-Ci-4 alkyl, carboxycycloalkyl, Ci-4-C 1-4 alkoxy and cyclic alkyl ether; A is a biologically active agent; Z is selected from the group consisting of: phosphate, phosphate ester, - (C = O) O-, carboxylic acid ester, thioester, amide, disulfide, amine, -NH (R1) -, amidine, hydrazone, -N = CH-, -NH-CH2-, , R ' -N = C- , -NH-C (Ri) H-, -S-CH2-C (OH) (R2) -, -O-CH2-C (OH) (R2) -, -C (= O) O-CH2-C (OH) (R2) -, - NR2-CH2-C (OH) (R2) -, -S- CH2-C (SH) (R2) -, -O-CH2-C (SH) (R2) -, -C (= O) O-CH2-C (SH) (R2) -, -NR2-CH2-C (SH) (R2) -, .R2 - N = C— -C (R2) H-NH-, - (C = O) -NH- (C = O) -NH-, R2 -C = N- (C = 0) -NH -C (R2) H-NH- (C = O) -NH-, - (C = O) -NH- (C = O) -O-, R; -c = N- (c = o) -a , -C (R2) H-NH- (C = O) -O-, - (C = O) -NH- (C = S) -NH-, 5 10 fifteen twenty 25 30 35 40 Four. Five R2 -C = N- (C = S) -NH , -C (R2) H-NH- (C = S) -NH-, - (C = O) -NH- (C = S) -O-, , -C (R2) H-NH- (C = S) -O-, image8 R2 -ON-NH , -NH- (C = O) -NH-, -O- (C = O) -NH-, -NH- (C = S) -NH-, -O- (C = S) -NH-, - S-CH2CH2- (C = O) -, -S-CH2CH (CH3) - (C = O) - -S-CH2CH2- (C = O) O-, -S-CH2CH (CH3) - (C = O ) O-, -S-CH2CH2- (C = O) NH-, -S-CH2-CH2- (pyridyl) -, -S-CH2-CH2- SO2-, -S-CH2-CH2-SO2-, - S-CH2- (C = O) -O-, -S-CH2- (C = O) -NH-, -S-CH2- (C = O) -, and image9 R1 is C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms; and R2 is H, C1-6 alkyl, C3-6 cycloalkyl, or an aryl group having 5-8 endocyclic atoms. 8. The compound according to claim 6 or claim 7, wherein E is bromine. 9. The compound of claim 6 or claim 7, wherein Sp1 is selected from the group consisting of a bond and image10 10. The compound of claim 6 or claim 7 wherein Z is independently selected from the group consisting of -NH-, -N = CH-, -NH-CH2 - and .R1 N-C- 11. The compound of any one of the preceding claims, wherein the phosphorylcholine-containing polymer has a molecular weight between about 0.5 kDa and about 200 kDa. 12. The compound of any one of the preceding claims, wherein the phosphorylcholine-containing polymer has a molecular weight between about 5 kDa and about 40 kDa. 13. The compound of any one of the preceding claims, wherein the phosphorylcholine-containing polymer has a molecular weight between about 10 kDa and about 20 kDa. 14. The compound of any one of the preceding claims, wherein said biologically active agent further comprises a Sp2 group. 15. The compound of any one of the preceding claims, wherein the biologically active agent is a protein. 5 10 fifteen twenty 25 30 35 40 Four. Five fifty 55 60 65 16. The compound of claim 15, wherein the biologically active agent is a therapeutic protein. 17. The compound of claim 15, wherein the protein is a human protein. 18. The compound according to claim 17, wherein the human protein is obtained by heterologous gene expression in a cell selected from the group consisting of a bacterium, a yeast cell, a cultured mammalian cell, a cell of insect in culture, a plant cell in culture, an avian cell in culture, a cell of a transgenic bird, a cell of a transgenic mammal, and a cell of a transgenic plant. 19. The compound of claim 17, wherein the human protein is obtained by gene activation in a cell line. 20. The compound according to any one of claims 1 to 14, wherein the agent is biologically active is selected from the group consisting of a cytokine, an enzyme, an antibody and a fragment of antibody. 21. The compound according to claim 20, wherein the cytokine, the enzyme, the antibody and the antibody fragment are human. 22. The compound according to claim 20 or 21, wherein the cytokine, the enzyme and the antibody or antibody fragment are obtained by heterologous gene expression in a cell selected from the group consisting of a bacterium, a yeast cell , a mammalian cell in culture, an insect cell in culture, a plant cell in culture, an avian cell in culture, a cell of a transgenic bird, a cell of a transgenic chicken, a cell of a transgenic quail, a cell of a transgenic goat, a cell of a transgenic cow, and a cell of a transgenic plant. 23. The compound according to any one of claims 1 to 14, wherein the agent is biologically active is selected from the group consisting of factor VIII, factor removed from domain b VIII, factor VIIa, factor IX, anticoagulants; hirudin, alteplase, tpa, reteplasa, tpa, tpa - 3 out of 5 domains removed, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogues, hgh, glucagones, tsh, follitropin-beta, fsh, gm- csf, pdgh, ifn alfa, ifn beta, ifn alfa2, ifn alfa2a, ifn alfa2b, ifn alfa2c, inf-apha1, ifn beta, inf-beta 1b, ifn-beta 1a, ifn-gamma, il-2, il-11 , hbsag, ospa, murine mab directed against the t-lymphocyte antigen, murine mab directed against tag-72, tumor-associated glycoprotein, fab fragments derived from chimeric mab directed against the platelet surface receptor gpM (b) / MI (a) , murine mab fragment directed against the tumor associated antigen ca125, murine mab fragment directed against the human carcinoembryonic antigen, cea, murine mab fragment directed against human cardiac myosin, murine mab fragment directed against the psma tumor surface antigen, murine mab fragments (fab / fab2 mixture) directed against hmw-maa, fragment d and murine mab (fab) directed against carcinoma-associated antigen, mab (fab) fragments directed against nca 90, a non-specific cross-reaction granulocyte surface antigen, chimeric mab directed against the cd20 antigen found on the surface of lymphocytes b, humanized mab directed against the alpha chain of the il2 receptor, chimeric mab directed against the alpha chain of the il2 receptor, chimeric mab directed against tnf-alpha, humanized mab directed against an epitope on the surface of the respiratory syncytial virus, humanized mab directed against her 2 , human epidermal growth factor receptor 2, human mab directed against the antigen associated with anti-ctla 4 cytokeratin tumor, infliximab gemtuzumab ozogamicin, ranibizumab, mab directed against the surface antigen of 20 dornase-alpha dnase lymphocyte, beta glucocerebrosidase, tnf-alpha, il-2-diphtheria toxin fusion protein, tnfr-lgg lar fragment fusion protein onidase, dnaasas, alefacept, darbepoietin alfa (colony stimulating factor), tositumomab, murine mab, alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (lggl), anakinra, biologic human modifier, nestriide (hbnp), colony stimulating factors, pegvisomant, human growth hormone receptor antagonist, recombinant activated protein c, omalizumab, immunoglobulin blocker e (lge), ibritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone , pigment hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic releasing factors, ethanorcept, antidiuretic hormones, prolactin, thyroid stimulating hormone, monoclonal antibody anti-HER2 humanized, anti-glycoprotein IIl-IIIa monoclonal antibody, antibody , murine cell surface antigen IgG2a antibody a nti-17-IA, murine anti-idiotype IgG antibody (GD3 epitope), chimeric anti-EGFR IgG antibody, humanized anti-aVp3 integrin antibody, humanized anti-CD52 IgG 1 antibody, humanized anti-CD33 IgG antibody, anti-IgG1 anti-CD33 antibody Chimeric CD2O, humanized anti-CD22 IgG antibody, humanized anti-ICAM3 antibody, primate anti-CD80 antibody, murine anti-CD20 antibody, humanized anti-CD40L antibody, primate anti-CD4 antibody, primate anti-CD23 antibody, IgG humanized anti-CD3, humanized anti-complement factor 5 (CS) antibody, humanized anti-TNF-a antibody, humanized anti-TNF-a Fab fragment, primate anti-CD4 IgG 1 antibody, human anti-CD4 IgG antibody, humanized anti-TNF-a IgG4 antibody, humanized anti-a4p7 antibody, humanized anti-CD4 IgG antibody, humanized anti-CD40L IgG antibody, humanized anti-VLA-4 IgG antibody, human anti-TGF-p2 antibody, anti receptor antibody -EGF (EGFr) monoclonal, mon antibody Human oclonal anti-VEGF, agalsidase, alefacept, aspariginase, amdoxovir (DAPD), antide, becaplermin, botulinum toxin, including types A and B and lower molecular weight compounds with botulinum toxin activity, calcitonins, cyanovirin, denileucine diftitox, erythropoietin 5 10 fifteen twenty 25 30 35 40 Four. Five fifty 55 60 65 (EPO), EPO agonists, alpha dornase, erythropoiesis stimulating protein (NESP), coagulation factors such as Factor V, Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, von Willebrand factor; ceredasa, cerezima, alpha-glucosidase, collagen, cyclosporine, alpha defensins, beta defensins, desmopressin, exendin-4, cytokines, cytokine receptors, granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), alpha inhibitor -1 proteinase, elcatonin, granulocyte colony stimulating factor (GM-CSF), fibrinogen, filgrastim, growth hormones, human growth hormone (hGH), somatropin, growth hormone releasing hormone (GHRH), GRO-beta , GRO-beta antibody, bone morphogenic proteins such as bone morphogenic protein 2, bone morphogenic protein 6, OP-1; acid fibroblast growth factor, basic fibroblast growth factor, CD-40 ligand, heparin, human serum albumin, low molecular weight heparin (LMWH), alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, consensus interferon; interleukins and interleukin receptors such as interleukin-1 receptor, interleukin-2, interleukin-2 fusion proteins, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6 , interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17 receptor; lactoferrin and lactoferrin fragments, luteinizing hormone releasing hormone (LHRH), insulin, pro-insulin, insulin analogues, amylin, C-peptide, somatostatin, somatostatin analogs, including octreotide, vasopressin, follicle stimulating hormone (FSH), imiglucerase, vaccine against influenza, insulin growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF), plasminogen activators such as alteplase, urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, and teneteplase; Neural growth factor (NGF), osteoprotegerin, platelet-derived growth factor, tissue growth factors, transforming growth factor-1, vascular endothelial growth factor, leukemia inhibitor factor, keratinocyte growth factor (KGF), factor of glial growth (GGF), T lymphocyte receptors, CD molecules / antigens, tumor necrosis factor (TNF) (e.g., TNF- and TNF-p), CTLA4, CTLA4 receptor, Fusion CTLA4-Fc, chemoattractant protein 1 monocytes, endothelial growth factors, parathyroid hormone (PTH), glucagon peptide, somatotropin, thymosin alpha 1, rasburicase, thymosin inhibitor alpha 1 IIb / IIIa, thymosin beta 10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase compounds (PDE), VLA-4 (very late antigen-4), inhibitors of VLA-4, bisphosphonates, respiratory syncytial virus antibody, cystic fibrosis transmembrane regulatory gene (CFTR), deoxyribo nuclease (Dnase), bactericidal / permeability enhancer protein (BPI), and anti-CMV antibody, etanercept (a dimeric fusion protein consisting of the extracellular ligand binding portion of the 75 kD human TNF receptor bound to the Fc portion of IgG1), abciximab, adalimumab, afelimomab, alemtuzumab, antibody against B lymphocyte, atlizumab, basiliximab, bevacizumab, biciromab, bertilimumab, CDP-484, CDP-571, CDP-791, CDP-860, CDu-860, CDu-860, CDu-860 , clenoliximab, daclizumab, eculizumab, edrecolomab, efalizumab, epratuzumab, fontolizumab, gavilimomab, gemtuzumab ozogamicin, ibritumomab tiuxetan, infliximab, inolimomab, keliximab, labetuzumab, lerdelimumab, olizumab, lym-1 radiolabelled metelimumab, mepolizumab, mitumomab, muromonad-CD3, nebacumab, natalizumab, odulimomab, omalizumab, oregovomab, palivizumab, pemtumomab pexelizumab, rhuMAb-VEGF, rituximab, satumomab pendetide, sevirumab, siplizumab, tositumomab, I131 tositumomab, trastuzumabirbumab, trastuzumabirbumab , tacrine, memantine, rivastigmine, galantamine, donepezil, levetiracetam, repaglinide, atorva statina, alefacept, tadalafil, vardenafil, sildenafil, fosamprenavir, oseltamivir, valaciclovir and valganciclovir, abarelix, adefovir, aminotron, amipropropyl, aminopropyl amine, aminopropyl amine, aminopropyl amidoproxamino sodium, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amlodipine, amsacrine, anagrelide, anastrozole, aprepitant, aripiprazole, aminosalicylic acid, asparaginase, atazanavir, atomoxetine, anthracycline, bexarotine, bibothrometin, bicalbotholbine, bicalbomotholbine, bibotoplazine, bicalbomine, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibetin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bibotin, bomatopyrol, bomotor, brometin , carmustine, cephalexin, chlorambucine, cilastatin sodium, cisplatin, cladribine, clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cis-retinoic acid, all-trans retinoic acid; dacarbazine, dactinomycin, daptomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, doxorubicin, dutasteride, eletriptan, emtricitabine, enfuvirtide, eplerenone, epirubicin, estramustine, ethinyl estradiol, etoposide, exemestane, ezetimibe, fentanyl, fexofenadine, fludarabine, fludrocortisone , fluorouracil, fluoximasterone, flutamide, fluticazone, fondaparinux, fulvestrant, gamma-hydroxybutyrate, gefitinib, gemcitabine, epinephrine, L-Dopa, hydroxyurea, icodextrin, idarubicin, ifosfamide, imatinib, itineconazole, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinoterazide, leinotezolidene , levamisole, lisinopril, lovotiroxina sodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, memantine, mercaptopurine, mequinol, bitartrate metaraminol, methotrexate, metoclopramide, mexiletine, miglustat, mitomycin, mitotane, mitoxantrone, modafinil, naloxone, naproxen, nevirapine, nicotine , nilutamide, nitazoxanide, nitisinone, norethindrone, octreotide, oxaliplatin, palonosetron, pamidronate, pemetrexed, pergolide, pentostatin, pilcamicina, porfimer, prednisone, procarbazine, prochlorperazine, ondansetron, palonosetron, oxaliplatin, raltitrexed, rosuvastatin, sirolimus, streptozocin, pimecrolimus, sertaconazole, tacrolimus, tamoxifen, tegaserod, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, tiotropium, topiramate, topotecan, treprostinil, tretinoin, valdecoxib, celecoxib, rofecoxib, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, voriconazole, dolasetron, granisetron, formoterol, fluticasone, leuprolide, midazolam, alprazolam, amphotericin B, podophyllotoxins, nucleosidase antivirals, aroyl hydrazones, sumatriptan, eletriptan; macrolides such as erythromycin, oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin, flurithromycin, dithromromycin, josamycin, spiromycin, midecamycin, loratadine, desloratadine, leucomycin, mithyamycin, swithiamycin; fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin, 5 10 fifteen twenty 25 30 35 40 amicacin, kanamycin, neomycin, and streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin, colistimethate; polymyxins such as polymyxin B, capreomycin, bacitracin, penems; penicillins, including penicillinase sensitive agents such as penicillin G, penicillin V; penicillinase resistant agents such as methicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, naphcillin; active agents of gram negative microorganisms such as ampicillin, amoxicillin, and hetacillin, cilia, and galampicillin; antipseudomonas penicillins such as carbenicillin, ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporins such as cefpodoxime, cefprozil, ceftbutene, ceftizoxime, ceftriaxone, cephalothin, cefapirin, cephalexin, cefradrine, cefoxitin, cephamandol, cefazolin, cephaloridin, cefaclorus, cephadroxyl, cephaloximatrin, cephaloximethyl, cephalophatrin cephaphthiophyte, cephalophatrin cephaphthiophyte cefoperazone, cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams such as aztreonam; and carbapenems such as imipenem, meropenem, and ertapenem, pentamidine isethionate, albuterol sulfate, lidocaine, metaproterenol sulfate, beclomethasone diprepionate, triamcinolone acetamide, budesonide acetonide, salmeterol, ipratrium trichlorate, chroma trichlorate of ergotamine; taxanes such as paclitaxel; SN-38, thyrostost and mimetics thereof. 24. The compound according to any one of claims 1 to 14, wherein said biologically active agent is selected from the group consisting of: erythropoietin, granulocyte colony stimulating factor (G-CSF), alpha interferon, beta interferon, Human growth hormone, and imiglucerase. 25. The compound of claim 24, wherein the biologically active agent is human erythropoietin. 26. The compound of claim 24, wherein the biologically active agent is the human granulocyte colony stimulating factor. 27. The compound of claim 24, wherein the biologically active agent is interferon alpha. 28. The compound of any one of claims 25 to 27, wherein the biologically active agent is an avian derivative. 29. The compound of claim 28, wherein the biologically active agent is a chicken derivative. 30. A pharmaceutical composition comprising a compound according to any one of claims 1 to 29. 31. A method of increasing the biological half-life property of a biologically active agent, comprising covalently binding a polymer containing branched phosphorylcholine to a biologically active agent, wherein the polymer has two or more polymer arms extending from a single group, in which the polymer portion of the compound is poly [2- (methacryloxyethyl) -2'- (trimethylammonium) ethyl] phosphate. 32. A compound according to any one of claims 1 to 29, or a pharmaceutical composition as defined in claim 30, for use in a method of treating the human or animal body by therapy.